► Serotonin is differentially involved in allocentric and egocentric working memory. ► Prefrontal serotonin depletion does not affect allocentric working memory. ► Prefrontal serotonin depletion impairs egocentric working memory. Working memory is a cognitive ability chiefly organized by the prefrontal cortex. Working memory tests may be resolved based on allocentric or egocentric spatial strategies. Serotonergic neurotransmission is closely involved in working memory, but its role in spatial strategies for working memory performance is unknown. To address this issue, prefrontal serotonin depletion was induced to adult male rats, and three days after the behavioral expression of both allocentric and egocentric strategies were evaluated in the “Y” maze and in a crossed-arm maze, respectively. Serotonin depletion caused no effects on allocentric-related behavioral performance, but lesioned rats performed deficiently when the egocentric working memory was evaluated. These results suggest that serotonin may be more closely related with the organization of working memory that uses own movement-guided responses than with that involving the use of external visuospatial signals. Further neurochemical studies are needed to elucidate possible interactions between serotonergic activity and other neurotransmitter systems in the organization of working memory-related allocentric and egocentric strategies.