On Earth-65, Spider-Woman Gwen Stacy has become increasingly isolated from her friends and estranged from her father George. George is hunting Spider-Woman for killing Peter Parker, unaware that it was an accident after Peter turned into the Lizard. Gwen encounters a version of the Vulture from an Italian Renaissance–themed alternate universe, and Miguel O'Hara and Jess Drew arrive through dimensional portals to help neutralize him. George corners Gwen and she reveals her identity to him, and he attempts to arrest her before she leaves with Jess and Miguel.
In Brooklyn on Earth-1610, 16 months after Kingpin's defeat and the destruction of the Alchemax collider, Miles Morales encounters the Spot, an Alchemax scientist-turned-supervillain whose body was infused with portals during the explosion. The Spot blames Miles for causing his mutation, and accidentally transports himself into a void and learns to use his portals to travel other universes. Gwen travels to Miles' dimension and they reconnect, sharing their struggles and hinting at feelings of attraction towards each other.
Gwen secretly tracks the Spot, and opens a portal to the next alternate universe he has gone to, Earth-50101. Miles discretely follows Gwen into the portal, and the two team up with Earth-50101's local Spider-Man Pavitr Prabhakar, and later Hobie Brown, against the Spot. The Spot absorbs the power of the collider, and he and Miles share a vision of his future attacks, which include the deaths of Jeff and Police Inspector Singh, the father of Pavitr's girlfriend Gayatri. The Spot escapes and Miles saves Singh from the collider's destruction, but the world begins to collapse before other Spider-People arrive to contain the damage.
Miles, Gwen, and Hobie are taken to Nueva York in Earth-928, where a multiversal society of Spider-People, known as the Spider-Society, work together to combat threats. Miles meets Miguel, the leader of the Society, and reunites with Peter B. Parker, who introduces him to his infant daughter Mayday Parker. Miguel explains to Miles that each of the Spider-People's stories contain "canon events", such as the death of a police captain close to Spider-Man, which cannot be disrupted otherwise they risk the collapse of that universe, which in turn will collapse the whole multiverse as a consequence.
Miles realizes that the Spot killing Jeff is a canon event and is imprisoned by Miguel to prevent Jeff from being saved. Miles frees himself with help from Hobie, and flees with the Society in pursuit. Miguel corners Miles and tells him that he was never supposed to become Spider-Man and that Earth-42 has no Spider-Man because of him. Miles overpowers Miguel and, with help from Margo Kess, flees back to his own universe. Gwen stands up for Miles, and is angrily ejected to her own universe by Miguel.
At home, Gwen reunites with George and they reconcile. Gwen begins searching for Miles, as do other members of the Society. Miles and Gwen realize that the former is in Earth-42 and not his own earth. In Earth-42, Miles' late uncle Aaron Davis is alive, and New York is crime-ridden due to the lack of Spider-Man. Aaron restrains Miles, and is joined by Miles' Earth-42 counterpart, the Prowler. As the Spot begins his attack on Earth-1610's New York, Miles prepares to escape his restraints. Gwen teams up with Peter B., Mayday, Pavitr, Hobie, and others.