Larry Hama was also the writer of the file cards written on the back of the packaging of the G.I. Joe toy line. The files where short biographies of both the Joes and Cobra soldiers.

The line of action figures included one for Zartan the Swamp Master, a villain who was a master of disguise and who could camouflage himself like a chameleon. The action figure had photoreactive "skin" that would turn blue when exposed to direct sunlight, as did his vehicle. In the animated series, Zartan was allergic to sunlight and would be weakened when exposed to it, which also turned his skin blue. However, the figure was taken off the shelves late in 1986.

Plans were made to have the character Rocky Balboa join the G.I. Joe team in 1987. In fact, Marvel Comics "G.I. Joe: Order of Battle" issue #2 contained a dossier for Balboa and Hasbro designed a prototype figure with Sylvester Stallone's likeness. Additionally, a new character, Big Boa, was added to the Cobra roster, complete with boxing gloves, to act as Rocky's Cobra counterpart and foil. A deal could not be reached to use Stallone's likeness, however, and Marvel wound up retracting the Balboa dossier, instead deciding on Sgt. Slaughter to be G.I. Joe's trainer and "celebrity spokesman".

Writer and script editor Buzz Dixon had developed a story titled "The Most Dangerous Man in the World" which explored the origins of Cobra and introduced the previously unseen founder of Cobra, a political theorist who laid out the groundwork for the organization but was shunted aside by Cobra Commander. Before the script got past the outline stage, Hasbro informed Dixon that the second season was to feature the new character of Serpentor, the Cobra Emperor, and Dixon's idea was scrapped. He did however release his 'alternate history' as a downloadable book in 2016.

Real-life Chicago Bears football star William "The Refrigerator" Perry became a member of the G.I. Joe team in late 1986, with the codename "Fridge." A special edition action figure was issued, but "Fridge" was never featured on the animated series.

Another large difference between the comic book and the cartoon is that fact that Storm Shadow switched sides and became a Joe in the comic, but remained loyal to Cobra throughout the initial run of the cartoon.

Sergeant Slaughter, a real-life professional wrestler, became a member of the G.I. Joe team during the show's second season. The real Sgt. Slaughter provided the voice for the animated character. The animated Sgt. Slaughter remained a member of the G.I. Joe team in later seasons, even after his real-life counterpart became an anti-American "heel" wrestler, teaming up with an "Iraqi General," General Adnan, against Hulk Hogan at Wrestlemania VII during the time of the 1990 Persian Gulf War.

At one point, Cobra was going to be Soviets, but this idea was dropped because it was considered culturally insensitive. Regardless, in "The Great Alaskan Land Rush," the Soviet counterpart to GI Joe, The October Guard, is an antagonist attempting to get an important seal in order for the USSR to reclaim Alaska.

Despite Larry Hama writing the file cards and the comic books, many of the characters depicted in the cartoon are vastly different than those in the file cards and the comic books.

There are many notable differences between the comic books and the cartoon. One particular difference is the rivalry between Spirit and Storm Shadow in the cartoon, where as in the comic the rivalry was between Snake Eyes and Storm Shadow.

Major Bludd's helmet resembles the helmet worn by Darth Vader in the Star Wars movies.