Riddler does seem the most likely option of the major Batman villains, but I think there's also a place for a creepy and dark take on the Mad Hatter if they're prepared to go down the route that the comic Hatter took in recent years. Perhaps a subplot? A spree of bizarre Riddler killings becoming entangled with a child-kidnap Mad Hatter theme?
I don't think they'd go with any of the more supernatural or goofy "deformed" villains; can't see Poison Ivy or Killer Croc making it somehow. I suppose if they want to keep drawing on the darker Batman comic material, they could always adapt the Holiday killings from "The Long Hallowe'en" somehow, which would also have the advantage of furthering the Batman-cops-Mafia triangle set-up that the first two movies have been built on.
I just hope they keep it hard and dark as hell; the Nolan/Bale movies have been some of the best comic book adaptations ever. I'd rank them up there with Superman 1&2, easily.