Subspecific affinities, determination of population boundaries, and levels of population connectedness are of critical importance for the development of management and conservation planning. We used variation at a mitochondrial locus and 5 biparentally inherited nuclear loci to determine partitioning of genetic variation of western big-eared bats (Corynorhinus townsendii) within and among caves occurring in a fragmented landscape of gypsum deposits in western Oklahoma. To accomplish this objective, we first performed a phylogenetic analysis based on the mitochondrial locus of western big-eared bats from a large portion of their range. This analysis indicated that western big-eared bats at the periphery of the distribution in western Oklahoma share phylogenetic affinities with the most geographically restricted subspecies, C. t. pallescens. Because C. townsendii is rare in Oklahoma and is listed as a species of special concern, this finding provides additional support for the continued protection of this species in Oklahoma. Within western Oklahoma, we failed to detect significant differentiation among any caves for the biparentally inherited microsatellite data. However, the mitochondrial locus exhibited significant levels of genetic differentiation among caves, with the highest level of differentiation occurring between caves within the disjunct distributions of gypsum (Φ_ST = 38.76%). Although a significant amount of genetic differentiation was detected between populations on the 2 disjunct distributions of gypsum deposits, Analysis with the program Migrate suggested high levels of asymmetric gene flow among some populations. Our results provide a greater understanding of the population dynamics of western big-eared bats on the periphery of their range and highlight the importance of continued monitoring and study of this taxon.