Before I begin, I got to say, Arkham City is my favorite video game of all time. I was pretty hyped when this game was announced. My only big problem is that I have to buy a PS4 to play it. That's extremely frustrating considering I just had to get a 3DS to play Arkham Origins Blackgate a few months back. Also, am I the only one surprised we didn't get any Arkham Origins tie-ins in the Batman: Arkham Unhinged comic book series? I think the series was cancelled, so we can't expect any Arkham Knight tie-ins but the series had a lot of untapped potential. A pity it was cancelled.
I did a condensed version of this list on Youtube (not in video format, though)in response to someone's video (using my Dark Knight 2149 Youtube account) but I decided to do an article on this because I thought this may be something the general fan community would want to read. Here is a breakdown of the villains we can expect to appear in the game. These are just speculations from what we've seen in Batman: Arkham Asylum and Arkham City.
1. AZRAEL

OK, so maybe he's not a straight up villain but it's safe to say
his small role in Arkham City was very import in setting up this game. The version we saw in Arkham City wasn't Jean Paul Valley (the most well known Azrael...to me, at least) but was a former cop named Michael Washington Lane. In AC, he gave an extremely important prophecy: "I bring a message: dark days are coming, Batman. The Prophecy is coming true, you are the warrior who will close the gates of Hell, you are the one who will save this day, and in doing so, events will occur that you cannot stop, from the ashes of Arkham the fires will rage and Gotham will burn, and you, you will burn too." It seems pretty obvious that this will have some effect on Arkham Knight.
2. ARKHAM KNIGHT
Thought this was just a random title for the game? So did I until it was revealed that this would be a villain in the game. Never heard of him? Thats because he is being created specificly for the game by Rocksteady (Batman Arkham Trilogy) and fan favorite comic book writer Geoff Johns (Batman: Earth One).
3. SCARECROW
Confirmed. I remember reading somewhere in a synopsis that Scarecrow will assemble a new roster of villains to take on The Batman including Two-Face, Penguin, and Harley Quinn. Anyways, in Arkham City it was revealed that Scarecrow had survived his apparent death in Arkham Asylum and is seemingly planning something big (which we caught a glimpse of in the trailer and by his voice over). Scarecrow is being funded by the Falcone Crime Family and is testing his formula on Arkham inmates. Encrypted messages (presumably by Scarecrow)can be heard in the form of numbers in Arkham City using the Cryptographic Sequencer. Scarecrows shredded mask can also be found and fear toxin canisters are hidden in one of the Riddler hostage rooms. In Arkham Asylum, it was implied Scarecrow may know Batman's identity.
4. THE FALCONE CRIME FAMILY
I honestly believe the Falcones are the main villains in the game. They may not have physically appeared in Arkham City but they definitly played a role. Heres what we know from riddles, easter eggs, ETC: The Falcones weren't in Arkham City because they left Gotham to avoid becoming prisoners. Outside of Gotham, they were funding Scarecrow and sending him supplies. The Falcones seem to be connected to a lot of villains in the game. It is stated in the game that much of the family didn't want to leave Gotham because the Falcones don't run from anything. Their leader, The Roman, made the discision to move. To me, The Roman doesn't seem like the type of villain to just run away. Is he planning something?
5. THE MARONI CRIME FAMILY
It is revealed in Arkham City via unlocks and easter eggs that the Maroni's are in a gang war with the Falcones.
6. THE BROKER
The Broker owns and sells property to villains. In Arkham City, he sold property to The Joker, Riddler, Zsasz, Great White Shark (according to Arkham Wiki, at least), Scarecrow, and Black Mask (according to Arkham Wiki, at least). He may or may not work for The Falcones.
7. CATWOMAN
In Arkham City, Catwoman is at war with Two-Face (who will be in Arkham Knight). In Arkham City, an incarcerated Calender Man asked Catwoman to free him from his cell. She replies, "Not after that Falcone mess." What is she refering to, I wonder? Could it have something to do with the Holiday Killer? Hmmmm.
8. CALENDER MAN
'Nuff said.
9. TWO-FACE
Appeared in Arkham City with a grudge against Catwoman. Confirmed to be in Arkham Knight. If I am not mistaken, I think he's one of the villains assembled by Scarecrow. In a patient interview tape in Arkham City, Two-Face reveals that the criminal who threw acid at his face in court wasn't Sal Maroni (who threw the acid in the comics), but actually a Falconi (most likely the Roman). Not only that, but he says that he's greatful to Falcone. Again, hmmm.
10. THE RIDDLER
The Riddler was easily one of the best things about Arkham City, which was my all time favorite Riddler story and incarnation. No Arkham game would be complete without The Riddler *flips middle finger at Arkham Origins Blackgate* and he has been confirmed to return in Arkham Knight.
11. THE RATCATCHER
In Arkham Asylum, The Ratcatchers equipment can be found in the ventilation system. In Arkham City, it is revealed that (after solving a riddle) that the Ratcatcher was caught stealing supplies from The Penguin and was never seen again (is he dead?). Because Ratcatcher's fate is unknown (and he IS a villain from the comics) and has been referenced in both Arkham games, I will be surprised if he's not in Arkham Knight. What makes him a scary villain to face is that, in the previous games, the ventilation systems are safe zones. The Ratcatcher can go into the vents (or send rats) WITH you and you have no way of defending yourself.
12. THE GREAT WHITE SHARK
The Great White Shark's detached nose, lips and ears can be found in a jar in the morgue (if I remember correctly, I think it was the morgue). In Arkham City, a building owned by the Shark, labeled "W White" after his real name (Warren White), can be seen. Because he was referenced in both games, I have no doubt he'll be in Arkham Knight.
13. HUSH
In Arkham Asylum, his name (Thomas Elliot) can be found on a dry erase board. He's a surgeon who apparently works at Arkham in this universe. In Arkham City, a killer goes around murdering inmates. He takes a piece of facial skin from each of his victums. When Batman discovers Hush's hideout, Hush removes the bandages from his face, revealing that he not only cut off his own face (kind of reminds me of The Dollmaker) but used the flesh he stole to create a new one for himself. The victums were hand picked because they each had a part of their face that resembled Bruce Wayne. Hush had a grudge against the Wayne family, so he made himself look nearly identical to Bruce Wayne as step one of his much larger plan. He escapes The Batman, but it is implied that his plan would unfold in a sequel. That sequel seems to be Arkham Knight. Unlike his comic book counter part, Hush doesn't seem to know The Batman's identity.
14. THE VENTRILOQUIST
Not only does his puppet, Scarface, physically appear in both games but The Ventriloquist himself is also mentioned in both games. Both also appear in Arkham Unhinged. I will be surprised if Rocksteady doesn't do something with the character.
15. THE JOKER?
I believe there is a 50/50 chance that the Joker will be ressurected in this game (especially after a CADMUS easter egg in Arkham City). Since the Joker is currently dead in the comics, following Death Of The Family (or at least thats what we're supposed to think), they'll probably ressurect him in the comics at the same time he's ressurected in the game (if he's ressurected in the game). Remember how Superman got married in the comics at the same time he got married in the Lois & Clark TV series? Same concept. Of course, I'm just speculating.
16. THE PENGUIN
Appears in the trailer.
17. HARLEY QUINN
Playable character via DLC who appears in the trailer.
UPDATE: 18. CLAYFACE
As Tainted87 pointed out in the comments, Clayface should appear because he fell into the Lazarus Pit in Arkham City. We don't know what effect this had on the character, especially considering he's a creature made of clay.
Did I miss anything? Disagree with anything? Let me know below!