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 (δD_p) derived from 2 geographic information system (GIS) models or latitude (LAT) and δD values in bat hair (δD_h), and examined intra- and interspecific variation in δD_h 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 δD_h than δD_p derived from either of the GIS-based models, although 1 model of δD_p performed better for some species. We found significant intraspecific differences in the relationships between δD_h and LAT and between δD_h and estimates of δD_p derived from Bowen et al. (δD_pB) for L. borealis, and significant variation in the regression equations of δD_h and LAT and δD_h and δD_p 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 δD_h values suggests that pooling across sex and age categories and use of surrogate species should be avoided.