Habitat use and feeding behaviors of cryptic animals are often poorly understood. Analyses of stable isotope ratios in animal body tissues can help reveal an individual's location and resource use during tissue growth. We investigated variation in stable isotope ratios of 4 elements (H, C, N, and S) in the hair of a sedentary species of insectivorous bat (Eptesicus fuscus) inhabiting a chemically complex urban landscape. Our objective was to quantify population-level isotopic variation and test for evidence of resource specialization by individuals. Bats were sampled over 3 annual molt cycles at maternity roosts in buildings and variance components analysis was used to test whether intraindividual isotopic variation among molts differed from interindividual variation, after controlling for year and roost-group effects. Consistent with prior evidence that E. fuscus is opportunistic in its habitat use and foraging at the population level, we observed wide population-level variation for all isotopes. This variation likely reflects the chemical complexity of the urban landscape studied. However, isotopic variation among years within marked individuals was lower than variation among marked individuals within year for all isotopes, and carbon signatures indicated resource specialization by roost groups and individuals. This is the 1st study to examine variation in stable isotope ratios of individual wild bats over multiple years. Although our results suggest this population tends toward opportunistic habitat use or prey selection, or both, during molt periods, results also indicate that individuals and groups of bats composing the population might be habitat or dietary specialists—a novel finding for insectivorous bats.