Stable-isotope analysis can address fundamental questions about the ecology and life history of mobile organisms. The hydrogen isotope, deuterium (δ²H), follows distinct and predictable patterns along environmental gradients, enabling migratory-origin assignments in vagile animals such as bats. However, it is unclear to what degree deuterium levels vary within non-migratory bat populations in fixed areas. To understand this, we compared deuterium signatures among adult female bats in maternity colonies of non-migratory Mid-Atlantic Eptesicus fuscus (Big Brown Bat). We sampled from 5 different locations along the Delmarva Peninsula. We also compared differences in signatures between males and females within colonies. Despite relative proximity to each other, deuterium signatures of females among colony locations differed significantly. Deuterium signatures did not differ among males and females or between 2010 and 2011. We suggest that variation in foraging and roosting behavior, as well as changes in water sources along the peninsula influence deuterium levels on a small geographic scale. These and other factors should be considered when interpreting sampled deuterium levels.