We analyzed population subdivision and gene flow of the Southeast Asian house mouse (Mus musculus castaneus) in Taiwan by using six microsatellite DNA markers. Seven populations of the house mouse (187 individuals), including one from Fukien Province in southeastern China, which is separated from Taiwan by the Taiwan Strait, were analyzed in this study. The overall polymorphic level at the six loci was high (H_e=0.76) although individual populations varied in their levels of heterozygosity (H_e=0.35–0.83). For the populations within Taiwan, there was no evidence of isolation by distance and the level of gene flow was not (inversely) correlated to geographic distances. Gene flow was estimated to be higher across the Taiwan Strait than within the island of Taiwan. These observations of gene flow cannot be understood unless in the context of the historical human settlements and agricultural expansion, and the commensal habits of the species. We also discussed the causes of population subdivision and genetic variation among populations in terms of ecological characteristics of the house mouse in Taiwan.