Narm Charm: The musical intro where the Joes take out Cobra on Liberty Island from blowing up the Statue of Liberty where the over-the-top action, the ridiculousness of Cobra's attack, and the cheesiness of the song ("Crashing through the sky! Comes a fearful cry! Of Cobraaaaa! (Cobraaaaaaaaa!) Cobraaaaaa! (Cobraaaaaaaaaaa!)") makes it looks like The War on Terror: The Musical. It's also beautifully animated, high tempo action, and a perfect distillation of the cartoon series in a single action sequence.
Nightmare Fuel: All of the Cobra-La stuff: Pythona and her break in to the Terrordrome, Cobra Commander turning into a snake, and the flashback to his original deformity, to Golobulus' plan to mutate all of humanity, especially the Imagine Spot of what would happen if it succeeds.
Signature Scene: The opening sequence. G.I. Joe foils a Cobra attack on the Statue of Liberty, with almost every single character who had ever appeared on the show contributing. Throw in Awesome Music and great moments like Duke punching Cobra Commander off the Statue.