Using mitochondrial and microsatellite DNA data and a population genetic approach, we tested male-mediated gene flow in the toad-headed lizards Phrynocephalus przewalskii. The mitochondrial DNA (ND2 gene), on the one hand, revealed two major lineages and a strong population genetic structure (FST = 0.692; FST′ = 0.995). The pairwise differences between the two lineages ranged from 2.1% to 6.4% and the geographical division of the two lineages coincided with a mountain chain consisting of the Helan and Yin Mountains, suggesting a historical vicariant pattern. On the other hand, the nuclear microsatellite DNA revealed a significant but small population genetic structure (FST = 0.017; FST′ = 0.372). The pairwise FST among the nine populations examined with seven microsatellite DNA loci ranged from 0.0062 to 0.0266; the assignment test failed to detect any naturally occurring population clusters. Furthermore, the populations demonstrated a weak isolation by distance and a northeast to southwest clinal variation, rather than a vicariant pattern. A historical vicariant event followed by male-mediated gene flow appears to be the best explanation for the data. Approximately 2–5 Ma, climatic change may have created an uninhabitable zone along the Helan-Yin mountain chain and initiated the divergence between the two mitochondrial lineages. With further climatic changes, males were able to disperse across the mountain chain, causing sufficient gene flow that eventually erased the vicariant pattern and drastically reduced the population genetic structure, while females remained philopatric and maintained the mitochondrial DNA (mtDNA) divergence. Although polygyny mating system and female philopatry may partially contribute to the reduced movement of females, other hypotheses, such as female intrasexual aggression, should also be explored.