The taxonomic status of the subspecies of Aselliscus stoliczkanus (Mammalia: Chiroptera: Hipposideridae) in China has yet to reach a consensus. To explore the variation and differentiation among populations of A. stoliczkanus from different regions of China, we conducted a series of bat surveys from 2012 to 2015. We performed multivariate morphological analyses using 16 external and 18 skull measurements and compared the sequence divergences of 2 mitochondrial genes (cytochrome b [Cytb] and cytochrome oxidase I [COI]). There were significant differences in external and skull measurements, particularly in the upper and lower canines, in the specimens from Guangxi compared with those from southwestern Yunnan and Guizhou. Sequence variation of the Cytb and COI genes was 11.0–13.2% and 12.4–14.0%, respectively, which represents differentiation at the species level. The samples from Guangxi were clustered with A. dongbacana from Vietnam in the phylogenetic trees based on COI sequences, and their sequence divergence was less than 3.2%. Based on the morphological and molecular results, we inferred that the samples from Guangxi were A. dongbacana, which represents a new record in China. Skull measurements of A. stoliczkanus from southwestern Yunnan exhibited significant morphological differences compared with those from Anlong County of Guizhou, and the sequence divergences of the Cytb and COI genes were 5.7–6.3% and 6.4–6.7%, respectively. Therefore, A. stoliczkanus from southwestern Yunnan and Anlong County of Guizhou might represent different subspecies. Finally, the phylogenetic analyses and minimal Cytb divergences of 0.2–0.9% indicated that specimens from southwestern and central Yunnan should be considered the same subspecies. Samples from Anlong County of Guizhou, Sichuan, and South Yunnan, with low Cytb divergences of 0.1–0.7%, represent another subspecies. A greater COI gene divergence was found between Libo and Anlong County of Guizhou; thus, the samples from Libo might represent yet another subspecies.