Twenty-five hibernating male Myotis lucifugus were collected from each of three abandoned mines in Ontario during November 1970. They were compared morphologically using forearm length and body weight and genetically using electrophoretic analysis of hemoglobin, indophenol oxidase, malate dehydrogenase, lactate dehydrogenase, succinate dehydrogenase, glucose-6-phosphate dehydrogenase, and tissue esterases. No significant difference in forearm size was observed, but differences in body weights of the bats from different populations were significant and proportional to the elapsed time between collection and processing. No variation within or between samples was observed for hemoglobin and indophenol oxidase. One population contained a single specimen homozygous for a rare allele at the malate dehydrogenase locus. Polymorphism was found to be the same in each population for lactate, succinate, and glucose-6-phosphate dehydrogenases. Great individual variation was present in the tissue esterases, but they yielded no evidence indicating genetic differentiation among the three populations. These results confirm that the M. lucifugus of Ontario consist of one large interbreeding population which is maintained, at least in part, by peregrinations of bats during swarming behavior.