The Amur sleeper Perccottus glenii (Perciformes, Gobioidei, Odontobutidae) is well known as an invasive fish in the river basins of Eastern and Central Europe, but its genetic background is unavailable across its native habitats in northeast Asia. In this study, we used the mitochondrial cytochrome b gene by sampling 19 populations of P. glenii across its native distributional areas of Liaohe and Amur River basins to explore its evolutionary history. Phylogenetic analyses identified three major clades within P. glenii, among which Clade A and Clade B were co-distributed in the Liaohe and Amur River basins, and Clade C was restricted to the latter. Molecular dating showed that the splits of Clades A, B and C have happened in the late Early-early Middle Pleistocene and the most recent common ancestors of these clades have been presented in the late Middle-early Late Pleistocene. The P. glenii showed very high levels of genetic structure among populations (ΦST = 0.801), probably due to the characters of its life histories with very limited dispersal ability. The admixture of different clades in some populations of P. glenii probably reflects historical secondary contact. These findings indicate that Pleistocene climatic oscillation and river capture were major determinants for genetic variations and evolutionary history of the P. glenii.