The endangered Ozark big-eared bat (Corynorhinus townsendii ingens) is restricted to eastern Oklahoma and western and north-central Arkansas, where populations may be susceptible to losses of genetic variation due to patchy distribution of colonies and potentially small effective population sizes. We used mitochondrial D-loop DNA sequences and 15 nuclear microsatellite loci to determine population connectivity among Ozark big-eared bat caves. Assessment of 7 caves revealed a haplotype not detected in a previous study (2002–2003) and gene flow among colonies in eastern Oklahoma. Our data suggest genetic mixing of individuals, which may be occurring at nearby swarming sites in the autumn. Further evidence of limited gene flow between caves in Oklahoma with a cave in Arkansas highlights the importance of including samples from geographically widespread caves to fully understand gene flow in this subspecies. It appears autumn swarming sites and winter hibernacula play an important role in providing opportunities for mating; therefore, we suggest protection of these sites, maternity caves, and surrounding habitat to facilitate gene flow among populations of Ozark big-eared bats.