This study examines the population structure and taxonomy of Tor douronensis, an important indigenous freshwater fish species in Malaysian Borneo, by using sequence analysis of 466 base pairs of the mitochondrial cytochrome c oxidase I (COI) gene. A total of 62 fish samples were collected from five locations in Sarawak (N=54) and Sabah (N=8). The phylogenetic analysis using the Neighbour-Joining (NJ) method supported the monophyletic status between T. douronensis and Tor tambroides, which further reinforced their taxonomic status as distinct species. The T. douronensis haplotypes were further divided into three major groups, with the Pelian fish from Sabah forming its own group (Cluster III) with strong bootstrap support. The large genetic differences separating the Sabah haplotypes from its Sarawak congeners suggested that the Pelian fish might represent a cryptic species. The current study showed high levels of intra and inter-population variations in T. douronensis. Within all population variations, T. douronensis populations were found, except in Bario. The presence of fixed haplotype differences along with high FST values among the populations of T. douronensis, support the conclusion that little or no migration occurred among the extant populations separated by large geographic distances or river systems. However, the sharing of haplotypes between some such populations, for example between Batang Ai and Bario (HS6), and between Batang Ai and Ulu Limbang/Ba Kelalan (HS2) provided support that T. douronensis had a historically widespread natural distribution in the region probably during the Quaternary period. Overall, the present study was able to shed light on the taxonomy and population structure of T. douronensis in Malaysian Borneo.