Rafinesque's big-eared bats (Corynorhinus rafinesquii) and southeastern myotis (Myotis austroriparius) are uncommon throughout their range and are listed (informally) by the U.S. Fish and Wildlife Service as species of management concern. Much of the decline in numbers of these species can be linked to a loss of bottomland hardwood forests throughout their range, resulting in a loss of natural roost structures in many areas. We characterized day roosts of both species and determined differences in characteristics of roost trees in western Tennessee. Radio transmitters were attached to adult females of both species to aid in locating new roosts. Thirty-one roost trees were identified, with most (93.5%) being large hollow water tupelos (Nyssa aquatica). Only two roost trees were used by both species during the survey period and no simultaneous roosting was documented. Roost trees in our study typically had triangular-shaped basal openings and large diameters (129.2 ± 7.3 cm). A significant difference (P < 0.05) in roost-tree diameter was observed between trees used by the two bat species. Our study is the first to compare characteristics of tree roosts of sympatric populations of Rafinesque's big-eared bats and southeastern myotis. Results suggest that these species may have different preferences for roost trees.