Abstract Background Global climatic oscillations, glaciation cycles and the unique geographic topology of China have profoundly influenced species population distributions. In most species, contemporary distributions of populations cannot be fully understood, except in a historical context. Complex patterns of Pleistocene glaciations, as well as other physiographic changes have influenced the distribution of bat species in China. Until this study, there had been no phylogeographical research on Myotis davidii, an endemic Chinese bat. We used a combination of nuclear and mitochondrial DNA markers to investigate genetic diversity, population structure, and the demographic history of M. davidii. In particular, we compared patterns of genetic variation to glacial oscillations, topography, and environmental variation during the Pleistocene in an effort to explain current distributions in light of these historical processes. Results M. davidii comprises three lineages (MEP, SWP and SH) based on the results of molecular variance analysis (AMOVA) and phylogenetic analyses. The results of a STRUCTURE analysis reveal multi-hierarchical population structure in M. davidii. Nuclear and mitochondrial genetic markers reveal different levels of gene flow among populations. In the case of mtDNA, populations adhere to an isolation-by-distance model, whereas the individual assignment test reveals considerable gene flow between populations. MDIV analysis indicate that the split of the MEP and SWP/SH lineages, and from the SWP and SH lineages were at 201 ka BP and 158 ka BP, respectively. The results of a mismatch distribution analysis and neutrality tests indicate a population expansion event at 79.17 ka BP and 69.12 ka BP in MEP and SWP, respectively. Conclusions The complex demographic history, discontinuous extant distribution of haplotypes, and multiple-hierarchy population structure of M. davidii appear associated with climatic oscillations, topography and eco-environmental variation of China. Additionally, the three regions are genetically differentiated from one another in the entire sample set. The degree of genetic differentiation, based on the analysis of mtDNA and nDNA, suggests a male-mediated gene flow among populations. Refuges were in the MEP, SH and the lower elevations of SWP regions. This study also provides insights for conservation management units (MEP, SWP and SH).