Here, we present a comprehensive analysis of the biogeographic history of Chinese house mice based on data from mtDNA control region sequences and 10 microsatellite loci in 535 individuals sampled from 29 localities. Phylogenetic analyses revealed two distinct evolutionary lineages corresponding to M. m. castaneus in the South and M. m. musculus in the North, with the Yangtze River roughly representing the boundary between these subspecies. More detailed analyses using published sequence data from mice sampled in neighboring countries revealed the migration routes of the two subspecies into China: M. m. castaneus appears to have migrated through a southern route (Yunnan and Guangxi), while M. m. musculus entered China from Kazakhstan through the northwest border (Xinjiang). Bayesian analyses of mitochondrial sequences indicated rapid population expansions in both subspecies, approximately 4,650-9,300 years ago for M. m. castaneus and approximately 7,150-14,300 years ago for M. m. musculus. Interestingly, the migration routes of Chinese house mice coincide with the colonization routes of modern humans into China, and the expansion times of house mice are consistent with the development of agriculture in southern China and northern China respectively. Finally, our study confirmed the existence of a hybrid zone between M. m. castaneus and M. m. musculus near the Yangtze River. Further study of this hybrid zone will provide a useful counterpart to the well-studied hybrid zone between M. m. musculus and M. m. domesticus in central Europe.