In this study, we report novel data on mitochondrial DNA in two of the largest eastern Bantu-speaking populations, the Shona from Zimbabwe and the Hutu from Rwanda. The goal is to evaluate the genetic relationships of these two ethnic groups with other Bantu-speaking populations. Moreover, by comparing our data with those from other Niger-Congo speaking populations, we aim to clarify some aspects of evolutionary and demographic processes accompanying the spread of Bantu languages in sub-Saharan Africa and to test if patterns of genetic variation fit with models of population expansion based on linguistic and archeological data. The results indicate that the Shona and Hutu are closely related to the other Bantu-speaking populations. However, there are some differences in haplogroup composition between the two populations, mainly due to different genetic contributions from neighboring populations. This result is confirmed by estimates of migration rates which show high levels of gene flow not only between pairs of Bantu-speaking populations, but also between Bantu and non-Bantu speakers. The observed pattern of genetic variability (high genetic homogeneity and high levels of gene flow) supports a linguistic model suggesting a gradual spread of Bantu-speakers, with strong interactions between the different lines of Bantu-speaker descent, and is also in agreement with recent archeological findings. In conclusion, our data emphasize the role that population admixture has played at different times and to varying degrees in the dispersal of Bantu languages. Am J Phys Anthropol, 2009. © 2009 Wiley-Liss, Inc.