This study quantified the intra‐specific diversity and characterized the population structure of the Asian snakehead fish, Channa striata in Sabah, North Borneo by determining the variability at six microsatellite loci. Range of genetic diversity across all sampled populations was comparatively moderate in relation to levels reported for C. striata populations occurring elsewhere and the overall Sabah population is highly structured, reflecting isolations across geological and ecological time scales. Two reciprocally monophyletic genodemes were identified along the west and east coast that may have been separated by mountain upthrusts throughout the central region of Sabah. Despite kinship among populations within each genodeme, individual demes were discrete as indicated by significant genotypic differentiation (all P < 0.0014) and low estimates of gene flow between them that likely reflect natural fragmentation of freshwater habitats they occupy. Our findings underline the potential of molecular markers in delimiting and delineating geospatial units of conservation in Sabah. Lowland freshwater ecosystems in the area may comprise two geographically isolated ecoregions, in which each hypothetically harbours biota that have evolved allopatrically. Additionally, identification of biodiversity hotspots within these putative ecoregions can be greatly facilitated with genetic‐level investigations as the rate by which freshwater communities are inventoried has been extremely slow in most habitats.