Background: Annual movements of tri-colored bats (Perimyotis subflavus) are poorly understood. While this species has been considered a regional migrant, some evidence suggests that it may undertake annual latitudinal migrations, similar to other long distance North American migratory bat species. Methodology/Principal Findings: We investigated migration in P. subflavus by conducting stable hydrogen isotope analyses of 184 museum specimen fur samples and comparing these results (δD fur) to published interpolated δD values of collection site growing season precipitation (δD precip). Results suggest that the male molt period occurred between June 23 and October 16 and 33% of males collected during the presumed non-molt period were south of their location of fur growth. For the same time period, 16% of females were south of their location of fur growth and in general, had not travelled as far as migratory males. There were strong correlations between δD fur from the presumed molt period and both growing season δD precip (males - r 2 = 0.86; p<0.01; females - r 2 = 0.75; p<0.01), and latitude of collection (males - r 2 = 0.85; p<0.01; females - r 2 = 0.73; p<0.01). Most migrants were collected at the northern (>40°N; males and females) and southern (<35°N; males only) extents of the species' range. Conclusions/Significance: These results indicate a different pattern of migration for this species than previously documented, suggesting that some P. subflavus engage in annual latitudinal migrations and that migratory tendency varies with latitude and between sexes. We suggest that this species' hibernation ecology makes it particularly susceptible to long winters, making migration from the northern extent of the species' range to more southern hibernacula preferable for some individuals. Fur δD values for some of the northern individuals may indicate an increase in the currently accepted northern range of this species. Sex-biased differences in migration may be the result of differences in reproductive pressures.