While Leonardo, Raphael, and Donatello all have brown eyes, Michelangelo has blue eyes. According to the film's director, this was done to make Michelangelo seem a bit more innocent and younger than the other turtles.
Some of the thirteen monsters are based on actual 'monsters' from popular myth - the first one that appears in the unfinished building is based on the Yeti, the next one to be captured is obviously the Cyclops, the one that eats a pigeon on a building is clearly modeled on a gargoyle, and the one in the diner brings to mind the Jersey Devil.
Karai's first movie appearance. In her original Mirage comic incarnation, Karai was completely unrelated to the Shredder and is actually a member of the Foot Clan's Council of Five, higher rank than the Shredder in the Foot Clan's global hierarchy. It is presumed Karai has the same status in this movie as she is not referred to as the adopted daughter of Shredder as she was in Teenage Mutant Ninja Turtles (2003).
During the fight between the Nightwatcher and the monster in the diner, at one point, a refrigerator falls on top of the monster and the music cuts out and the monster is heard making a whimpering noise. This was actually a mistake in the soundtrack, but when the filmmakers saw it, they couldn't stop laughing and left it in the film.
As revealed on the director's commentary on the DVD one of the early drafts of the script had Raphael briefly dying and Donatello using Winter's technology to bring him back to life. This idea was turned down by Peter Laird however who disliked the idea of having one of the turtles dying.
TMNT 2 would have loosely adapted the Turtles' 13-part comic book saga City At War: Michelangelo feels like the "odd man out" among his brothers, not being taken seriously. With the best of intentions, the character runs away and joins the Foot Clan, donning a black bandanna. Meanwhile, the Turtles travel to Japan and cross paths with Karai in an adventure resulting in the return of their arch nemesis, Shredder. TMNT 3 would have featured the Triceratons as well as the Technodrome's arrival from Dimension X, with Donatello being a catalyst for these events. Kevin Munroe wanted Michael Clarke Duncan to voice the Triceraton's leader, Commander Mozar. He even had poster designs already in mind for TMNT 2 ("a wet city street, by a manhole cover, Mikey's orange bandanna lying on the ground") and its sequel ("the Technodrome with the Turtles very small in the foreground").
Splinter's appearance is generally consistent with the previous movies (in which he is depicted as having brown fur), although in this movie, he wears a proper karate gi, as opposed to dirty rags from the previous movies. His right ear is also no longer cut as it was in previous movies. Kevin Munroe jokingly responded that Splinter had an otoplasty.
Jim Cummings is the only Teenage Mutant Ninja Turtles actor to make it into this movie since his first TMNT appearance in Teenage Mutant Ninja Turtles (1987), which he played Leatherhead, Genghis Frog, and one of the occasional voice alternates for Shredder.
Specific dialog makes references to the previous live-action films. In the battle with the Foot Clan, Casey Jones says, "Two minutes for high sticking!" which was one of his lines from his introduction in the same film. The very last line of the film, spoken by Raphael is, "Man, I love being a turtle!" which has been spoken in each of the previous films.
During the first Nightwatcher scene, when the criminals are fleeing, one of the signs on the shops in the background spells out "monro" in hiragana, a Japanese writing system, which is most likely related to the writer and director Munroe.
One sequel idea that had already been nixed by Peter Laird had the Turtles go back in time to explore Mayan culture, just as an evil alien race also arrives via teleportation. According to Kevin Munroe, Laird's response was, "Eh, we did it in 1992," a reference to Teenage Mutant Ninja Turtles III (1993).
The idea of April O'Neil knowing ninjitsu skills is taken from the Teenage Mutant Ninja Turtles (2003) animated series were she received some training from Master Splinter. Also the color of April O'Neil's yellow jumpsuit is a nod to and the original Teenage Mutant Ninja Turtles (1987) cartoon where she was a reporter wearing a yellow jumpsuit with pockets.
If the monster that attacked the diner is indeed based on the Jersey Devil as suggested, then it is rather appropriate that Kevin Smith provided the voice of the diner's chef, since he is an avid and loyal New Jersey Devils hockey fan.
This is the only incarnation in the franchise, out of all the television series and movies (not including Saban's Ninja Turtles: The Next Mutation (1997)) to not have the name "Teenage Mutant Ninja Turtles", instead using the initials, which fans usually use to shorten the name.
The trivia items below may give away important plot points.
The film is considered to some fans as a loose continuation of the original trilogy, ignoring the events of the television series Saban's Ninja Turtles: The Next Mutation (1997), but is really set in its own universe. Towards the end of the film, several items are shown that reference the live action movies. These include: The Shredder's helmet and staff, a Foot Ninja Mask, the canister of ooze that mutated the turtles and Splinter (labelled TCRI instead of TGRI, though a crack is strategically placed to work either way), the Time Sceptre, Walker's hat, Lord Norinaga's helmet, and pieces of the suits of armor worn by the Turtles in the third film.
The scene where Casey and Raphael are being attacked, Casey says "And I thought insurance salesmen were pushy!" This line was also used in Teenage Mutant Ninja Turtles (1990) by Michaelangelo when the Foot Clan was invading April's apartment.