Nucleotide sequences of 3' end of the cytochrome b gene, tRNA genes, D-loop control region, and the 5' end of the 12S rRNA of mitochondrial DNA (mtDNA) were used to assess the genetic and phylogeographic structure of Acrossocheilus paradoxus populations, a Cyprinidae fish of Taiwan. A hierarchical examination of populations in 12 major streams from three geographical regions using an analysis of molecular variance (AMOVA) indicates high genetic differentiation both among populations (PhiST = 0.511, P < 0.001) and among regions (PhiCT = 0.368, P < 0.001). Limited migration largely contributed to the genetic differentiation. High nucleotide diversity (1.13%) and haplotype diversity (0.80%) were detected among populations. The degree of genetic differentiation was correlated with geographical distance between populations, a result consistent with the one-dimensional stepping stone models. A neighbour-joining tree recovered by (DAMBE) supports the pattern of isolation by distance and reveals a closer relationship between populations of the central and southern regions. A minimum spanning network based on nucleotide substitutions reflected migration routes from populations of the central region to the northern and southern regions, respectively. Postglacial colonization and expansion can explain the phylogeographical pattern. Single and ancient migration events may have allowed the northern region to attain the monophyly of mtDNA alleles. In contrast, most populations within geographical regions are either paraphyletic or polyphyletic due to the relatively shorter time period for coalescence. Both low haplotype number and genetic variability suggest a bottleneck event in the Chingmei population of northern Taiwan. Based on coalescence theory, the monophyly of the Tungkang population of the southern region may be associated with a founder event.