The game's plot is largely unchanged from Metroid II. It now features a Super Metroid-style introduction narrated by Samus Aran (narration above), about the nature of her mission on SR388. Over the course of the game, Samus's scanners automatically download Logbook entries similar to the Metroid Prime series that reveal additional lore about areas and bosses. She can uncover optional areas that include the landing sites of the G.F.S. Thoth and an Anhur-class patrol ship carrying the two ill-fated Galactic Federation teams sent to rescue the Galactic Federation Special Squadron sent previously. Metroid II did not feature the final resting places of these Federation teams, despite mentioning them in the manual. Samus also encounters surviving Federation Marines battling a Zeta Metroid that metamorphoses into an Omega Metroid before effortlessly killing them. It is not possible to save the marines. The mysterious earthquakes linked to the deaths of Metroids are insinuated to be caused by the Queen Metroid herself, as her cries can be heard during the last few in the game. The Logbook reveals that the cave system is located between two tectonic plates on SR388. The game ends with Samus killing off the remaining Metroids, the Queen, and discovering an infant Metroid she later calls the baby.
After the credits, Samus speaks the famous introductory phrase from Super Metroid: "The last Metroid is in captivity. The galaxy is at peace." In the best ending, Samus is seen in her Gunship calling the Space Science Academy about the baby, leading into Super Metroid.