In an attempt to fill knowledge gaps relating to genetic structure in the endangered gray bat (Myotis grisescens), we investigated geographic patterns in multilocus microsatellite DNA (msDNA) genotypes and mitochondrial DNA (mtDNA) haplotype frequencies across eight primary hibernacula. Isolation-by-distance (IBD) was absent in the msDNA data and no bottlenecks were detected, with genotypic diversity (A_R = 6.52, H_o = 0.64) and overall genetic differentiation (F_ST = 0.024, P < 0.001) being comparable to other Myotis and North American vespertilionids. Genetic structure (Φ_CT = 0.063, P < 0.001) among groups of populations (K = 2) was observed. We also identified a pattern of slight east-west regional genetic structure, likely attributable to the natural barrier of the Mississippi River Alluvial Plain, in the haplotype data (Φ_CT = 0.086, P < 0.05), along with moderate IBD (r = 0.486, P < 0.05). Though genetic differentiation among populations was generally low, significant interpopulation genetic structure, likely arising from some degree of philopatric behavior and a lack of hibernacula-associated mating sites within the Mississippi River Alluvial Plain, was observed.