Columbia was probably the most different, followed by Challenger. Columbia was the heaviest by a good margin (8400 pounds), which made it inappropriate for ISS missions. Additionally, considerable improvements were made to the Thermal protection system. Columbia didn't have any of the silicon thermal blankets, but used white-covered tiles in medium temperature areas. I think that Challenger had some of the blankets, but not as many as the later orbiters (Discovery, Atlantis, and Endeavour), all of which had similar thermal protection system installations. Don't know about cockpit configurations. Columbia also had a camera on the top of the vertical stabilizer to take thermal images of reentry heating.
Discovery was always intended to be a flying orbiter. Enterprise was once intended to be converted into a flying orbiter, but it was decided that it was not worth it to convert it to orbital flying status after the glide tests. It never had real engines, a thermal protection system, and lacked many other systems not necessary for testing of the craft during its final descent (the 747 piggyback "drop" tests).
BTW, the next US space vehicle will not be reusable. The CEV is an expendable concept, that is part of NASA's plan to go to the Moon, and eventually Mars. It is based on non-reusable elements, which may not be so bad, as many of the Shuttle's more troublesome features (such as the tile-based and RCC-based thermal protection system, and the expensive and complex main engines) were due to reusability.