The transformation from a foraging way of life to a reliance on domesticated plants and animals often led to the expansion of agropastoralist populations at the expense of hunter-gatherers (HGs). In Africa, one of these expansions involved the Niger-Congo Bantu-speaking populations that started to spread southwards from Cameroon/Nigeria approximately 4,000 years ago, bringing agricultural technologies. Genetic studies have shown different degrees of gene flow (sometimes involving sex-biased migrations) between Bantu agriculturalists and HGs. Although these studies have covered many parts of sub-Saharan Africa, the central part (e.g. Zambia) was not yet studied, and the interactions between immigrating food-producers and local HGs are still unclear. Archeological evidence from the Luangwa Valley of Zambia suggests a long period of coexistence ( approximately 1,700 years) of early food-producers and HGs. To investigate if this apparent coexistence was accompanied by genetic admixture, we analyzed the mtDNA control region, Y chromosomal unique event polymorphisms, and 12 associated Y- short tandem repeats in two food-producing groups (Bisa and Kunda) that live today in the Luangwa Valley, and compared these data with available published data on African HGs. Our results suggest that both the Bisa and Kunda experienced at most low levels of admixture with HGs, and these levels do not differ between the maternal and paternal lineages. Coalescent simulations indicate that the genetic data best fit a demographic scenario with a long divergence (62,500 years) and little or no gene flow between the ancestors of the Bisa/Kunda and existing HGs. This scenario contrasts with the archaeological evidence for a long period of coexistence between the two different communities in the Luangwa Valley, and suggests a process of sociocultural boundary maintenance may have characterized their interaction.