Jessie operates within Team Rocket as part of a trio with James and Meowth. Her childhood involved poverty after her mother, the high-ranking Team Rocket agent Miyamoto, vanished during a mission to find Mew; food scarcity led Jessie to sometimes eat snow. She faced multiple career setbacks: failing to graduate from a Chansey-focused Pokémon nursing school (though befriending a Chansey that evolved into Blissey), receiving the lowest recorded scores at Pokémon Tech alongside James, involvement in a bicycle gang earning nicknames like "Big Jess" and "Chainer Jessie," and brief attempts as a model, ninja, and weather reporter. Before joining Team Rocket, she experienced failed romantic relationships and a rejected opportunity to become a Pokémon Coordinator with a boy named Astin.

In the Black and White series, Giovanni promotes Jessie, James, and Meowth to Advanced Agents, significantly altering their operations and demeanor. Jessie adopts a more focused, calm, and strategically calculated approach, temporarily shedding her short-tempered, dramatic nature. This includes wearing a charcoal-gray uniform and employing covert tactics like disguises. With increased resources and direct leadership backing, the trio prioritizes efficiency in pursuing rare Pokémon, particularly Ash's Pikachu, and participating in operations like the Meteonite plan.

During this arc, Jessie captures Unova-region Pokémon. She obtains a Woobat off-screen in a cave swarming with the species per Giovanni's directives; it frequently battles Ash's Pidove and Oshawott using moves like Gust and Air Slash. Later, she adds a Frillish, her first Ghost-type. These Pokémon exhibit less personal bonding and function primarily as mission tools. Both are relinquished to Giovanni in the series finale but returned to her in Journeys.

Core traits persist despite her temporary evolution. Jessie maintains intense vanity, especially regarding her magenta hair, reacting furiously to damage or insults. Her rivalry with fellow agent Cassidy remains a motivator. She continues participating in Pokémon performances like Contests and Showcases under aliases such as Jessilina, displaying ambition and skill without her usual villainous overreactions. After Operation Tempest is thwarted, Jessie reverts to her volatile, theatrically expressive personality, though the Advanced Agent promotion leaves a residual impact on her tactical confidence.