Taxonomic confusion among island flying foxes (Pteropus hypomelanus) still in debate as in the lack of further genetic studies that describe subspecies validity needs verification. On the basis of morphological variants, 17 subspecies of P. hypomelanus are currently recognized. A previous study showed the distribution of two subspecies of P. hypomelanus in Malaysia: P. hypomelanus robinsoni in Pangkor Island, west coast of Peninsular Malaysia and P. hypomelanus lepidus from Perhentian Island, Kapas Island, and Tioman Island, east coast of Peninsular Malaysia. Herein, using the displacement loop (D-loop) region of mitochondrial DNA sequences, phylogenetic and haplotype network was conducted on their population structure in Peninsular Malaysia upon 42 genetic samples from Dangli Island (Langkawi), Pangkor Island (Perak), Redang Island (Terengganu), and Tinggi Island (Johor). Phylogenetically, two main clades separating northwest and southeast populations emerged. No haplotype was shared between the populations. The results from genetic differentiation (estimate of population subdivision (FST) and nucleotide subdivision (Nst)) yielded a gene flow that was nearly cut-off because of the central mountain that forms as an effective barrier between islands of the east and west coasts of Peninsular Malaysia. Although the above finding genetically confirms their subspecies designation, examination using a nuclear marker is needed in future studies. [ABSTRACT FROM AUTHOR]