Mary Jane is depicted as an extremely beautiful, green-eyed redhead, and has been the primary romantic interest of Peter Parker for the last twenty years, although initially competing with others for his affection, most prominently with Gwen Stacy and the Black Cat. Mary Jane's relatively unknown early life was eventually explored in The Amazing Spider-Man #259.

Early issues of The Amazing Spider-Man featured a running joke about Peter dodging his Aunt May's attempts to set him up with "that nice Watson girl next door", whom Peter had not yet met and assumed would not be his type, since his aunt liked her (in the Parallel Lives graphic novel an identical scenario is shown between Mary Jane and her Aunt Anna). Mary Jane made her first actual appearance in The Amazing Spider-Man #25 (June 1965), although her face was obscured. It is not until The Amazing Spider-Man #42 (November 1966) that her face is actually seen. In that issue, on the last page, Peter finally meets her, and he is stunned by her beauty even as she speaks the now-famous line: "Face it, Tiger... you just hit the jackpot!