The turtles struggle to contain an outbreak of mutations that occurs thanks to the leftover mutagen from the thwarted Kraang invasion. Kirby is among the victims of the outbreak, and a misunderstanding leads April to become very upset and ashamed of the turtles and break off their friendship. However, the turtles are able to earn her forgiveness when they save her from Karai, who has taken temporary command of the Foot while the Shredder is away in Japan. Along with April came her new friend Casey Jones, who helps repel an assault on the turtles’ lair. Kirby is eventually restored to his human form when Donatello manages to concoct another Kraang chemical called retro-mutagen, which causes organic beings to reverse their major physical transformations, and help keep it away from former T.C.R.I inventor Baxter Stockman, who gets mutated into Stockman-Fly while under Shredder’s employ.
Meanwhile, Shredder returns from Japan with the mutated Japanese bounty hunter Tiger Claw as his new second-in-command. Tiger Claw is later sent through a portal to the 1987 cartoon universe, but returns.
During a battle with the turtles, Karai is informed of her true nature by Leonardo (who has developed a crush on her since season 1), but she is too reluctant to accept it. When she is taken to the lair, she finally realizes the truth and disowns Shredder, who responds by imprisoning her. Repeated attempts to free Karai ultimately succeed, but Shredder captures her again and uses her as bait to kill the Hamato Clan. However, Karai ends up mutated into a serpent, to everyone’s horror.
In response to this, Shredder has the Foot Clan help the Kraang, who have just perfected the previously unstable mutagen, launch a second invasion of New York, starting by destroying the turtles’ lair and forcing them to abandon it. The Kraang begin mutating New York’s populace, including Kirby, despite the efforts of Earth’s military and the turtles. Leonardo is gravely wounded when he is ambushed by the entire Foot, and Splinter is seemingly killed during a battle with Shredder. The turtles, April, and Casey are forced to flee to the O’Neil family’s summer home in Upstate New York as the Kraang successfully conquers Manhattan.