Mature forest is a key resource for hollow-using bats, but its importance in shaping where bats roost during breeding is not well understood. This lack of understanding limits the ability of forest managers to make informed decisions on the type, amount and spatial arrangement of mature forest to retain for bats in areas used for timber production. Using radio-telemetry, day roosts of three sympatric hollow-using bat species – the chocolate wattled bat (Chalinolobus morio), the Tasmanian long-eared bat (Nyctophilus sherrini) and the lesser long-eared bat (Nyctophilus geoffroyi) – were located in two forested landscapes in south-eastern Tasmania, Australia. By radio-tracking 24 bats in the maternity season, 76 roosts were located, with interspecific variation in roosting preferences evident at the roost, patch and landscape scale. Maternal colonies showed a clear selection for roosting in areas of the landscape containing the highest availability of mature forest, with smaller patches, strips and individual trees used to a greater extent for roosting in the landscape where mature forest was scarce. These findings showcase the importance of retaining mature forest at multiple spatial scales for hollow-using bats.