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. 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. 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 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, who explains to Miles that each of the Spider-People's stories contain "canon events" 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 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 flees back to his own universe.
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.