Protecting day roosts is imperative to the persistence and survival of local populations of tree-roosting bats. The Pine Ridge region of Nebraska represents the largest area of coniferous forests in the state and possesses the greatest diversity of bats in Nebraska. In this study, we examined roost characteristics primarily for 4 species in need of conservation in the region: Fringed Myotis (Myotis thysanodes), Long-legged Myotis (Myotis volans), Little Brown Myotis (Myotis lucifugus), and Hoary Bat (Aeorestes cinereus). With a limited understanding of roost characteristics for other species in the region, we also collected some roost data for the Big Brown Bat (Eptesicus fuscus) and Western Small-footed Myotis (Myotis ciliolabrum). We tracked 42 bats with an emphasis on lactating females. Fringed Myotis, Big Brown Bats, and Western Small-footed Myotis typically roosted in cracks and crevices of dead trees. Long-legged Myotis roosted only in rock crevices at our western-most study site and only large, dead Ponderosa Pines (Pinus ponderosa) at our eastern-most site. Little Brown Myotis roosted in human-made structures in large colonies. Hoary Bats roosted in foliage of live hardwoods and conifers. Conservation of dead Ponderosa Pines, particularly those with large diameters and exfoliating bark, will provide roost structures for most crevice-roosting bats in the region. Our limited data for Hoary Bats suggest that roost-tree use is variable, as individuals roosted in both live hardwoods and conifers. Exclusive use of buildings throughout the region by female Little Brown Myotis implies timber harvests and wildfires might not greatly affect roosts for reproductive females. We demonstrated that roost characteristics vary among species and between nearby populations of the same species, representing important information for making informed habitat management decisions in a region with limited forests in northwestern Nebraska.