In this study, the HLA-DRB1 and -DQB1 molecular diversity of two Berber-speaking populations of Southern Tunisia was analysed. Genetic comparisons indicate that both populations exhibit peculiar profiles for HLA-DRB1, as they diverge significantly from most other North Africans, while being highly diversified. At the opposite, they are much less differentiated from neighbouring populations according to the HLA-DQB1 polymorphism. Overall, the HLA class II genetic structure of Arab and Berber-speaking populations from Tunisia, and of North Africa as a whole, is complex and cannot be simply explained by geographic or linguistic differentiations. The present North African genetic pool has probably been shaped by both genetic drift and the contribution of genetically heterogeneous populations during the history of settlement of North Africa.