Roosts are an integral habitat component for species of bats and may affect their survival and fitness. Conversion of forests to agricultural and urban areas may decrease available roosting habitat for the endangered Indiana bat (Myotis sodalis). Furthermore, the effects of habitat loss could be exacerbated if sympatric species favor and compete for similar roost-sites. We used radio-telemetry to study roosting habits of two species of Myotis in northeastern Missouri. We did not directly test for competition between these species for maternity roosts; rather, our goal was to determine if similarities in roost site characteristics were strong enough to warrant further investigation into competition for maternity roosts. Of 118 roosts located throughout the study, 79 were used by Indiana bats and 39 by northern bats (M. septentrionalis). Differences in roost structure (natural vs. manmade), tree status (live vs. snag), roost type (bark vs. cavity/crevice) and canopy coverage indicate that subtle, but biologically important differences exist in roost selection. Northern and Indiana bats both relied heavily on trees as roosts sites; however, Indiana bats roosted in trees with lower canopy cover and less often in cavities and live trees than northern bats. Our results suggest that niche separation in roost selection exists between northern and Indiana bats.