The caterpillars of Gonimbrasia belina, commonly known as mopane worms, are intensively harvested for human consumption in southern Africa. Concerns over the sustainability of the species have been raised for the last two decades due to increasing demand, habitat erosion, and climate change. Despite its cultural, economic, and nutritional significance, genetic data for G. belina was largely unavailable. We used a multi-marker approach including mitochondrial sequences and nuclear ddRADseq data to assess the genetic diversity, phylogeographical structure and demographic history of G. belina in Namibia and the Limpopo River Basin (South Africa and Botswana). Mitochondrial sequences revealed strong phylogeographical structure at the broad scale separating Namibia and Limpopo River Basin populations. Within the Limpopo River Basin, populations are separated by the Limpopo River, with haplotype sharing only at the border between South Africa and Botswana. Mitochondrial genetic diversity varies between sampling areas but overall is highest in South Africa and lowest in Botswana, while historical demography points to population expansion in Namibia but not in South Africa and Botswana. Nuclear data in the Limpopo River Basin revealed some connectivity among populations albeit with significant genetic differentiation and unique gene pools in some sampling areas. All sampling areas show low genetic diversity, alarmingly small effective population size, and signs of recent bottlenecks. We generated the first baseline data for the genetic monitoring of G. belina populations and conclude that concerns over the sustainability of the species are presently justified in Botswana and South Africa.