Genetic variability generally is associated with adaptive potential of species and can be assessed using microsatellite markers. Mexican long-nosed bats (Leptonycteris nivalis) are endangered, migratory nectarivores thought to be experiencing population declines. Despite historical and current tracking efforts, migratory connections among roost sites remain elusive. Our objective was to assess the genetic variability and connectivity of two of the most geographically separated populations of L. nivalis currently known—a mating roost in central Mexico used from September to March, and a maternity roost in western Texas used by adult females and their young from June to August. Twelve loci developed for L. yerbabuenae and four for Glossophaga soricina amplified L. nivalis DNA; of those 16 loci, seven (all from L. yerbabuenae) were useful in genetic analyses of 113 individuals. High levels of genetic variation in L. nivalis from the two sites were not significantly different and no substructure was detected between these two roost sites separated by more than 1,200 km. Further, we recovered evidence of close relationship (parent–offspring) in nine pairs of juveniles captured at the northern roost and adults from the southern roost, confirming a connection between the two locations. For this endangered species, the level of variation detected, the lack of a recent bottleneck, and estimates of effective population size, are encouraging for future recovery. Management plans aimed at conserving Mexican long-nosed bats should recognize the need for managing these bats as a single population and conserving foraging and roosting habitat along migratory routes to reduce population fragmentation.