Until recently, the little brown bat (Myotis lucifugus) was a common bat species in North America. However, this species faces a significant threat from white-nose syndrome (WNS), which will likely result in the listing of M. lucifugus as a priority species in eastern parts of its range. The aim of this study is to examine the genetic structure of M. lucifugus, and thereby infer the impact that WNS is likely to have on M. lucifugus populations. Samples were collected from over 500 individuals from eastern hibernacula and from maternity colonies throughout the United States and Canada. Both mitochondrial (cytochrome oxidase I) and nuclear (14 microsatellites) loci were examined. Our data reveal restricted gene flow among females, but not males, from winter colonies. This mitochondrial genetic structure mirrors topographical variation across the region. Broader sampling of summer maternity colonies reveals a role for the Rocky Mountains as a historically significant, although not complete, barrier to gene flow among both males and females. Microsatellite data indicate that colonies in the Rockies provide opportunities for admixture between characteristically eastern and western lineages, suggesting that these mountains are unlikely to act as barriers to the westward spread of WNS across the continent. Our results will help us to place current patterns of mortality and disease spread in eastern North America in the broader context of population-level processes across the range of this species