The tricolored bat (Perimyotis subflavus) has experienced extensive declines in regional abundance — near 30% as of 2013 — due to disease, habitat loss, and anthropogenic conflict. There is urgency to determine whether the species should be protected in the United States; however, critical species-specific information required to make this decision is lacking. Here, we address those critical knowledge gaps by (i) estimating genetic diversity, and (ii) assessing population structure. We utilized sequence data from a 562 base pair region of the mitochondrial genome and eight autosomal microsatellite loci from 14 sampling locations across the core of P. subflavus’ range and found substantial diversity in both data types. Further, we found contrasting patterns of population structure between the data types. Weak evidence for population structure was detected using the mitochondrial data, with the most probable population break dividing eastern and western sampling sites, and evidence for isolation-by-distance across the sampled region. Conversely, we detected no evidence for structure or isolation-by-distance using microsatellite data, revealing a panmictic population. The findings reported here represent a baseline understanding of genetic diversity and structure for P. subflavus; additional analyses using samples collected post white-nose syndrome emergence are required to understand losses in genetic diversity due to recent population declines.