The phylogenetic relationships among 10 species of Rhinolophus and 10 species of Hipposideros from Borneo and Peninsular Malaysia were successfully inferred from the partial mitochondrial DNA (mtDNA) cytochrome (cyt) b sequences. Of the 413 nucleotide positions examined, there were 171 positions (41.4%), of which 164 positions (95.9%) were parsimoniously informative. The phylogenetic trees reconstruction using neighbour-joining (NJ), unweighted maximum parsimony (MP) and maximum likelihood (ML) methods suggest the monophyletic clustering of these families. The interspecific relationships within Rhinolophidae were completely resolved, while those within Hipposideridae were not fully resolved, as supported by the low bootstrap values. Overall, the phylogenetic analysis using partial mtDNA cyt b gene was useful to discriminate these complicated taxa and successfully revealed the misidentification of several specimens before due to their similar morphologies.