Molecular genetics tools have been used recently to investigate aspects of bat biology, including the degree of male- and female-driven gene flow among populations. However, none of this work has focused on the little brown bat (Myotis lucifugus), one of the most common bats in North America. Previous work using mark–recapture suggests that like many mammals, including other temperate vespertilionid bats, gene flow in this species is largely driven by males, whereas females return to the roosts in which they were born to rear pups. To determine if this species displays a pattern of population genetic structure that is consistent with this hypothesis I sampled 182 female M. lucifugus at 12 maternity colonies throughout Minnesota. Using a portion of the mitochondrial gene cytochrome b and 10 polymorphic nuclear microsatellites, I found significant structure among colonies, and population differentiation was slightly higher for the mitochondrial locus. This supports the hypothesis that female M. lucifugus display some degree of natal philopatry, but the inferred female dispersal rate was high. The pattern of structure was complex and demonstrates that unequal gene flow among maternity colonies is occurring and that the co-occurrence of breeding populations within maternity colonies and/or inbreeding could be confounding estimates of population genetic structure among these colonies. The patterns of population genetic structure and natal philopatry imply that both seasonal migration and dispersal by females should be considered as possible factors in the spread of emerging infectious diseases such as white-nose syndrome.