Houseflies (Musca domestica L., Diptera: Muscidae) are cosmopolitan, colonizing, and eusynanthropic. Their distribution in the Malaysian archipelago provides an opportunity to study successive waves of colonization and extinction during the Pleistocene and Recent epochs. We scored single‐strand conformation polymorphisms (SSCPs) at 16S2 and COII mitochondrial loci in 47 housefly samples from the Australian, Austro‐Malayan, Indo‐Malayan, Manchurian and Indo‐Chinese subregions of Wallace's zoogeographical classification. We discuss the results in light of the Pleistocene vs. post‐Pleistocene dispersal and faunal exchange in the Asia‐Pacific area. Fourteen haplotypes were detected, of which 10 were confined to a single subregion. No haplotype was ubiquitous and only one was found in four subregions. Population diversity, HS, was greatest in the Indo‐Malayan (0.36) and heterogeneous among subregions. The mean subregional diversity was 0.21 ± 0.03, representing the probability that two randomly chosen flies, from any subregion, had different haplotypes. The hierarchical partition of diversity indicated restricted maternal gene flow among subregions (GRT = 0.60, Nm∼ 0.32). These results suggest long‐standing genetic isolation of houseflies in the Malaysian archipelago and support the hypothesis that they dispersed widely during the Pleistocene. Haplotypes common among mainland populations but shared with island groups in low frequencies (<1%) indicate surprisingly little recent gene flow.