The genus Scotophilus is composed of 15 recognized species with 7 species distributed throughout sub-Saharan Africa, 4 distributed across southern and southeastern Asia, 3 endemic to Madagascar, and 1 endemic to Reunion Island. Scotophilus is plagued with problems in species definition, and systematic relationships among members of the genus are poorly understood. We used mitochondrial DNA (mtDNA) and Y-chromosome sequence data from 11 of the 15 recognized species, which represent the most comprehensive taxonomic coverage to date, to examine phylogenetic patterns within Scotophilus. All trees have S. kuhlii from Asia as the most basal species followed by S. nux from Africa. However, S. heathii from Asia is embedded within the other African Scotophilus, indicating a complex biogeography with multiple continental exchanges. Furthermore, the Malagasy taxa are most closely related to 2 different African species, suggesting independent colonizations of Madagascar from the continental mainland. In addition, African S. dinganii did not comprise a monophyletic group but exhibited at least 2 additional cryptic species based on high levels of genetic divergence in the cyotchrome-b gene. The large-bodied S. nigrita is closely related to S. dinganii with a similar mtDNA haplotype but distinct zfy haplotype, suggesting a possible hybridization event in the most recent common ancestor that potentially represents a mitochondrial capture. Overall measures of interspecific genetic distances ranged from 4.2% to 19.2% for mtDNA data and 0.18% to 2.14% for Y-chromosome data, indicating that members of the genus Scotophilus are highly divergent from one another.