The genetic subdivision of the endemic minnow, Zacco pachycephalus, in 12 rivers of Taiwan was studied by allozyme electrophoresis. Among 26 loci surveyed, six were significantly differentiated among sites. Allozyme genotype frequencies within samples accord with Hardy–Weinberg expectations. A highly divergent genetic structure among sites (FST = 0.497) with low genetic variability within samples (H = 0.006–0.062, mean = 0.03) suggests that local populations originated from a small number of founders. upgma and Fitch trees derived from Nei’s genetic distance and hierarchical analysis of FST values reveal that samples of Z. pachycephalus can be divided into northern, middle, and southern subgroups and that most genetic variation (87%) is distributed among rather than within groups. It is proposed that there were two isolated refugia during the most recent glacial period, and that populations of the southern group (Group I) originated from a southern refugium in Taiwan below the boundary of the Kaoping River, and other groups (Group II), including northern and middle subgroups, originated from a refugium located on the extinct landbridge between Taiwan and mainland China. The postglacial division between northern and middle subgroups may be due to hydrological differences between these two regions.