The desert locust, Schistocerca gregaria, is found in disjunct Southern and Northern areas of Africa. While the subspecies S. g. gregaria is a feared agricultural pest since early civilization, with plagues documented from West Africa to Southwest Asia, the anecdotic S. g. flaviventrisinhabit 2500 km Southern in the Namib and Kalahari deserts. Questions have been raised regarding the taxonomic distinctness of S. g. flaviventris and S. g. gregaria subspecies, which are morphologically very similar. However, fixed and diagnosable differences in traits under genetic control are a criterion for subspecies designation. In this study, we used DNA barcoding to delimit taxonomical entities by amplifying a standard 658-bp fragment of Cytochrome C Oxidase Subunit I gene (COI) and 28 versatile microsatellite markers to assess the evolutionary relationships of closely related populations or subspecies. COI sequences clustered into two monophyletic lineages concordant with geography. In addition, characteristic point mutations allowed a clear diagnose between the two subspecies. Microsatellite genotyping of 13 populations showed that the two taxonomic units were not united by gene flow. The levels of genetic divergence were 5 to 30 times greater between S. g. flaviventris and S. g. gregaria than within subspecies. Our research also suggested that mid-Holocene climatic changes triggered the present geographical distribution and diversification of the desert locust subspecies. Indeed, Approximate Bayesian Computation estimated a divergence time between subspecies of around 7000 years ago, which corresponds to the mid-Holocene, a time of greater humidity, where the vegetation cover in Africa was dominated by forest and closed vegetation landscapes. It is likely that between the drier last glacial maximum and the divergence time of the subspecies (a period of 18,000 years), a continuous arid range stretched from southwestern Africa to northern Africa. At mid- Holocene, deserts and g