The fragmentation of forests by the expansion of agriculture is recognized as an important factor influencing worldwide declines of forest-dependent species. Species that are forest dependent may be especially vulnerable to fragmentation because they have specialized resource requirements and may exhibit lower mobility in an agricultural matrix. We investigated movement patterns and resource selection of forest-dependent northern long-eared myotis (Myotis septentrionalis) in a forest–agricultural landscape on Prince Edward Island, Canada. Radiotelemetry was used to locate day-roosts and to estimate locations of female bats during nightly foraging bouts. Day-roost locations and foraging areas were mapped using a geographic information system to generally characterize the forest cover of foraging and roosting areas. Vegetative structure and insect prey availability were measured in the field and compared between foraging and roosting areas to describe resource selection at these sites. Movements of female northern long-eared myotis were constrained to forest features and foraging areas were concentrated along forest-covered creeks with bats roosting predominantly in deciduous trees within the same forest fragment, although bats at 1 site seemed to exclusively use a barn during late pregnancy and lactation. Differences in prey availability did not explain the spatial segregation of roosting and foraging areas. Relative to roost areas foraging sites were more likely to be close to forested creeks and densely forested areas, whereas roost sites were characterized by the availability of potentially suitable roosts. This study demonstrates the importance of investigating movements and resource selection of individuals in fragmented landscapes because a specialization on forest resources can highly restrict the vagility of forest-dependent species to a local environment.