Stable hydrogen isotopes (δDs) in metabolically inert tissues such as feathers and hair provide a set of endogenous markers that may be useful for establishing migratory connectivity in animals. We tested the assumption of a clear relationship between δD values of growing-season-weighted average precipitation (δDp) derived from 2 geographic information system (GIS) models or latitude (LAT) and δD values in bat hair (δDh), and examined intra- and interspecific variation in δDh of 4 bat species in the eastern United States. We analyzed 251 hair samples from 1 long-distance migrant (eastern red bat [Lasiurus borealis]) and 3 regional migrants (Indiana bat [Myotis sodalis], northern long-eared bat [M. septentrionalis], and little brown bat [M. lucifugus]) captured during the reproductive period (pregnancy and lactation) when bats are resident. LAT explained more of the variation in δDh than δDp derived from either of the GIS-based models, although 1 model of δDp performed better for some species. We found significant intraspecific differences in the relationships between δDh and LAT and between δDh and estimates of δDp derived from Bowen et al. (δDpB) for L. borealis, and significant variation in the regression equations of δDh and LAT and δDh and δDp among species for adult females and adult males. Stable hydrogen isotope analysis may be a valuable tool for studying migratory connectivity in bats, but significant intra- and interspecific variation in δDh values suggests that pooling across sex and age categories and use of surrogate species should be avoided.