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The prevailing view of the social organization of temperate bats is that maternity colonies represent aggregations of philopatric females connected to other maternity colonies via male-mediated gene flow. However, few studies have examined fine-scale patterns of matrilineal diversity within and between colonies, and clear evidence of strict philopatry generally is lacking. Here we examine patterns of genetic diversity within and among maternity colonies of big brown bats (Eptesicus fuscus) using both microsatellites and mitochondrial sequences. As in other temperate bats, we observed no differentiation among colonies using nuclear microsatellite markers (FST: −0.0001–0.012; 99.5% of variation within populations). Overall, there was significant mitochondrial structure among colonies, but the pattern was complex, with low Φst values among some populations and high values among others (range: −0.007–0.491). Three groups of colonies were identified in nested analyses of molecular variance, among which variation was high (21.2% of the variation), but within which variation was low (1.4%), and significant matrilineal isolation by distance (r = 0.56, P = 0.012) was observed. Maternity colonies were characterized by high haplotype diversity (h = 0.83) and a large number of matrilines (5–15) per colony. Furthermore, we observed a small number of maternities within colonies, and only 5–17% ( = 9%) of the sampled females were found roosting with a mother or daughter. Our results are inconsistent with strict female philopatry, and suggest that female-mediated gene flow may occur among some maternity colonies. The potential for female dispersal in temperate bats prompts new and exciting questions about social and dispersal dynamics.