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Mitochondrial DNA cytochrome b sequences (1141 bp) in 229 specimens of Acrossocheilus paradoxus from 26 populations were identified as four lineages. The pairwise genetic distances among these four lineages ranged from 1.57 to 2.37% (mean= 2.00%). Statistical dispersal-vicariance analysis suggests that the ancestral populations were distributed over mainland China and Northern and Western Taiwan. Approximate Bayesian computation approaches show that the three lineages in Taiwan originated from the lineage in mainland China through three colonization routes during two glaciations. The results indicated that during the glaciation and inter-glacial periods, the Taiwan Strait was exposed and sank, which contributed to the dispersion and differentiation of populations. Furthermore, the populations of A. paradoxus colonized Taiwan through a land bridge to the north of the Formosa Bank, and the Miaoli Plateau in Taiwan was an important barrier that limited gene exchange between populations on both the sides.