One of the most widely distributed bats in the New World, the big brown bat (Eptesicus fuscus) exhibits well-documented geographic variation in morphology and life history traits, suggesting the potential for significant phylogeographic structure as well as adaptive differentiation among populations. In a pattern broadly consistent with morphologically defined subspecies, we found deeply divergent mitochondrial lineages restricted to different geographic regions. In contrast, sequence data from two nuclear loci suggest a general lack of regional genetic structure except for peripheral populations in the Caribbean and Mexico/South America. Coalescent analyses suggest that the striking difference in population structure between genomes cannot be attributed solely to different rates of lineage sorting, but is likely due to male-mediated gene flow homogenizing nuclear genetic diversity across most of the continental range. Despite this ongoing gene flow, selection has apparently been effective in producing and maintaining adaptive differentiation among populations, while strong female site fidelity, maintained over the course of millions of years, has produced remarkably deep divergence among geographically isolated matrilines. Our results highlight the importance of evaluating multiple genetic markers for a more complete understanding of population structure and history.