Myotis sodalis (Indiana bat) and Myotis grisescens (gray bat) are federally endangered species in the United States that use a limited number of hibernacula, placing them at risk of extirpation and/or extinction from various threats. Understanding their genetic structure and diversity can help inform the management and preservation of their adaptive genetic variation, but prior genetic studies of these species were based on small numbers of microsatellites and mitochondrial DNA. Here, we used genotyping-by-sequencing (GBS) to assess the genetic population structure and effective sizes of M. sodalis and M. grisescens. We genotyped 146,548 single-nucleotide polymorphisms (SNPs) from 45 M. sodalis and 57,902 SNPs from 47 M. grisescens individuals spread across their ranges. Population structure for each species was assessed using ADMIXTURE and Principal Component Analyses (PCA) of GBS data. Myotis sodalis showed no geographic structure in genetic diversity, suggesting a panmictic population structure. Although there was also no evidence of subpopulations of M. grisescens, a PCA for M. grisescens indicated some east–west population variation and gene flow across the Mississippi River Valley. Coalescent modeling suggested an effective population size of 360,977 (95% Confidence Interval, CI 349,488–368,414) for M. sodalis and 41,888 (CI 41,400–243,122) for M. grisescens, with increasing population sizes for both across evolutionary timescales. From a genetic conservation standpoint, M. sodalis may best be managed as a single population, whereas M. grisescens should be managed with consideration of maintaining the genetic variation of both eastern and western groups.