Tim Drake's blonde wife is a nod to Stephanie Brown, who was Tim's girlfriend in the comics at the time. It was acknowledged in the DVD commentary of the movie.
Cross-Dressing Voices: Andrea Romano plays Young Tim Drake during his fits of laughter and is actually credited as "Laughing Boy" in the film. Matthew Valencia couldn't pull off the maniacal, Joker-like giggle that the producers wanted because his voice had changed too much.
Throw It In: The bit at the end with Nanna Harley wasn't originally in the script, but Dini didn't want to let the character go. Bruce Timm agreed to leave it in because the funny moment served as a good release after the climax.
Ghoul came about because Michael Rosenbaum's impression of Christopher Walken was never used in previous collaborations.
The salute the police officer gives Batman wasn't in any storyboard given to the animators. Everyone was surprised to see it in the final print, and found it too hilarious to be upset.
Too Soon: The reason there is both a cut and uncut version of this movie - the original release date for the uncut version was going to be on Halloween 2000◊, which was unfortunate when it was still during the fallout of the Columbine shootings, and movie companies were coming under heavy criticism for violence in films. As a result, the film was postponed, then heavily edited and released as the unrated version on December 12, 2000. Fortunately, the developers still retained the original version in shelves and eventually released it as "the original, uncut version" under the PG-13 rating on April 23, 2002 (just three days after the third anniversary of the Columbine tragedy) following an online petition to have it released.
What Could Have Been: The initial design for the Dee-Dee twins was very different◊ to the finalized film. Probably a good idea that they changed it, as it perhaps gave away the fact that they were Harley Quinn's granddaughters.
The comic includes several scenes that did not make it to either versions of the film, such as:
Bruce's visit to the remains of Arkham Asylum to find clues on the Joker's return. He is unknowingly followed by Terry. (Storyboard drawings, however, do appear as deleted scenes, which were present on both versions of the DVD as part of the special features.)
Batman's interrogation of the Penguin in the flashback. Both were part of the script that got cut out of the movie due to time and pacing concerns, as confirmed in the commentary.
The original ending was going to have it turn out the Joker actually was Tim Drake, with no microchip involved, but that didn't sit well with the test audience.
During the flashback, The Joker wears the "Kiss the Cook" apron in both the cut and uncut versions. Originally it was going to say "Kill the Cook", but the execs decided that the words would be too gruesome.
Word of God: The writers admitted that as they talked about the novel Hannibal one night, after the book came out but while the movie was being made, elements inspired by the novel description of Hannibal Lecter made their way into the Joker designs for the movie—the slicked-back hair on the future Joker and the red eyes on both the future and flashback/Justice League/Static Shock Joker designs.
Harley managed to survive her fall into the chasm at Arkham because of the serum Ivy gave her in "Harley & Ivy".