Many endangered species live in fragmented and isolated populations with low genetic variability, signs of inbreeding, and small effective population sizes - all features elevating their extinction risk. The flat-headed loach (Oreonectes platycephalus), a small noemacheilid fish, is widely across southern China, but only in the headwaters of hillstreams; as a result, they are spatially isolated from conspecific populations. We surveyed single nucleotide polymorphisms in 16 Hong Kong populations of O. platycephalus to determine whether loach populations from different streams were genetically isolated from each other, showed low levels of genetic diversity, signs of inbreeding, and had small contemporary effective population sizes. Estimates of average observed heterozygosity (H = 0.0473), average weighted nucleotide diversity (π = 0.0546) and contemporary effective population sizes (N = 10.2 ~ 129.8) were very low, and several populations showed clear signs of inbreeding as judged from relatedness estimates. The degree of genetic differentiation among populations was very high (average F = 0.668), even over short geographic distances (<1.5 km), with clear patterns of isolation by distance. These results suggest that Hong Kong populations of O. platycephalus have experienced strong genetic drift and loss of genetic variability because sea-level rise after the last glaciation reduced connectedness among paleodrainages, isolating populations in headwaters. All this, together with the fact that the levels of genetic diversity and contemporary effective population sizes within O. platycephalus populations are lower than most other freshwater fishes, suggests that they face high local extinction risk and have limited capacity for future adaptation.