To examine the phylogeographic relationships of Microtus fortis in China, we investigated 84 individuals collected from five populations. The mitochondrial cytochrome b gene (cyt b) and control region (CR) were sequenced and 49 haplotypes were observed. No shared haplotype was found among different geographic populations. High Fst values among the populations suggested that fragmentation of habitat has resulted in genetically distinct populations. The trees, inferred from maximum likelihood and Bayesian phylogenetic analysis, highly supported all the M. fortis individuals clustering into one monophyletic lineage. Three main clades are recovered within M. fortis: (1) North group; (2) South group; and (3) GX group. The North Group distributed on the north side of Qinling Mountains-Huaihe river line as well as the South Group was on the south. It suggests this geographic barrier played an important role in differentiation of M. fortis in China. Furthermore, the samples all from Southwest China in the GX group may be an example of ‘refuge within refugia’ in glacial period. According to our molecular clock analysis, the main clades of M. fortis divergence and separated time at around 0.77±0.64 million years ago (Mya) located in the Penultimate Glaciation. Divergences within the three clades taken place during the interglacial period between the Penultimate Glaciation and the Last Glaciation. Bayesian skyline plot indicates the effective population size of M. fortis had been experiencing a downward trend in the past decades, which may due to the habitats loss and environmental degradation.