Genetic variation and population structure of the leaf-nosed bat Hipposideros speoris were estimated using 16S rRNA sequence and microsatellite analysis. Twenty seven distinct mitochondrial haplotypes were identified from 186 individuals, sampled from eleven populations. FST test revealed significant variations between populations in the overall pairwise estimation (FST = 0.710; p < 0.001). In addition, haplotype network and analysis of molecular variation analysis (AMOVA) consistently suggest the prevalence of genetic structure in the sampled populations. However, the mtDNA data was not significantly different in few closely located urban populations, but significant difference has been observed with the use of microsatellite data. The Bayesian clustering analysis identified eight clusters among the populations; the clustering pattern also corresponded to the haplotype networks. Overall, the present study suggests a “macrogeographic genetic isolation-by-distance” and possibility of gene flow among closely located populations.Key words: mtDNA, 16S rRNA, microsatellite, population structure, Hipposideros speoris
