The Eastern Cape Province harbors almost half of the indigenous forest in South Africa, but these forests are threatened by large-scale agricultural and urban development planned for the coming decade. Additional anthropogenic development is likely to cause further fragmentation and degradation of forests inhabited by the dusky pipistrelle bat (Pipistrellus hesperidus). We used eight microsatellite markers to study the genetic diversity, population structure, and migration, of P. hesperidus (n = 120) across 14 sites in the Eastern Cape and KwaZulu-Natal provinces of South Africa. We examined the effect of contemporary land cover types on genetic differentiation to assess whether current levels of urbanization and agricultural development affect gene flow. High gene flow and low population structure were evident across sampled sites, apart from genetic discontinuities at the northern (Oribi Gorge Nature Reserve) and southern (Alexandria Forest) ends of the seaboard. Genetic discontinuity at Oribi Gorge may relate to anthropogenic modification of two rivers surrounding the forest, while the Alexandria-linked barrier is a climatic break known as the Bedford gap. Migration rates generally were low between sites except for one Scarp forest, Manubi State Forest, from which individuals dispersed to other sites. The Amatole Mistbelt forests supported high genetic diversity, and likely served as a refugium for P. hesperidus during the Last Glacial Maximum. The composition of land cover classes between sites was a poor predictor of genetic differentiation, although it seems likely that P. hesperidus uses riparian habitats and wetlands for dispersal. Lastly, urban and agricultural development did not have a significant effect on genetic differentiation, which may reflect the wide niche breadth and intermediate distribution range of the species. This study provides insights into genetic diversity and gene flow of P. hesperidus across the study region prior to agricultural intensification and large-scale urbanization.