An apparently disjunct population of tricolored bats (Perimyotis subflavus) exists in southwest Nova Scotia. During the summers of 2003–2004 and 2007–2008 we trapped bats in mist nets set at a bridge over the Mersey River in Kejimkujik National Park. In total we captured 42 tricolored bats (41 female and 1 male) and fitted 32 females and the single male with radio transmitters and tracked them to their roost sites daily. Characteristics of roost sites were compared to those of randomly selected, potentially available roost sites using an a priori—selected candidate set of models and ranked using AIC_C for model selection. In total we found 99 unique roost trees in 9 spatially distinct (8 female, 1 male) roosting areas. On average, bats spent 2.5 d in a roost tree. While conspecifics from other parts of the species' range typically roost in tree foliage, maternity colonies of tricolored bats in Nova Scotia roosted exclusively in Usnea trichodea lichen, typically in conifers (of which 91% were spruce; Picea spp.) that were on average 212 m from water. Only 4% of the 300 random trees from the landscape contained Usnea trichodea. These trees were a mixture of softwood and hardwood (58% and 42%, respectively) and were, on average, 550 m from water. These findings represent an apparently unique relationship between lichens and bats that likely impacts many aspects of the species biology, including distribution, social structure, and parasite prevalence.