Stomoxys calcitrans (Diptera: Muscidae: Stomoxyini), a synanthropic fly with a worldwide distribution, is recognized to have an important medical and veterinary impact. We conducted a phylogeographic analysis based on several populations from five major zoogeographic regions of the world in order to analyse population genetic structure of S. calcitrans and to trace its global dispersion. Results from mitochondrial (COI, Cyt-b and ND1-16S) and nuclear (ITS2) DNA show a substantial differentiation of Oriental populations (first lineage) from the Afrotropical, Palearctic, Nearctic, Neotropical and Oceanian populations (second lineage). The divergence time analyses suggest the separation between the two lineages approximately in mid-Pleistocene. Oriental populations are isolated and would not have participated in the colonization of other regions, unlike the Afrotropical one which seems to be the source of S. calcitrans dispersion towards other regions. Demographic analyses indicate that Oriental, Afrotropical and Palearctic regions have undergone a population expansion during late Pleistocene-early Holocene. The expansion time of this cosmopolitan species could have been influenced by continental human expansions and by animal domestication. Research highlights : We investigate population structure and demographic history of Stomoxys calcitrans. We find that S. calcitrans populations are partitioned into two lineages. The first lineage is Oriental whereas the second consists of remaining populations. Isolation between the two lineages is suggested to occur around mid Pleistocene. Expansion time for S. calcitrans populations is approximately 12,000–7000 years ago.