Abstract The expansion of people speaking Bantu languages is the most dramatic demographic event in Late Holocene Africa and fundamentally reshaped the linguistic, cultural and biological landscape of the continent 1–7 . With a comprehensive genomic dataset, including newly generated data of modern-day and ancient DNA from previously unsampled regions in Africa, we contribute insights into this expansion that started 6,000–4,000 years ago in western Africa. We genotyped 1,763 participants, including 1,526 Bantu speakers from 147 populations across 14 African countries, and generated whole-genome sequences from 12 Late Iron Age individuals 8 . We show that genetic diversity amongst Bantu-speaking populations declines with distance from western Africa, with current-day Zambia and the Democratic Republic of Congo as possible crossroads of interaction. Using spatially explicit methods 9 and correlating genetic, linguistic and geographical data, we provide cross-disciplinary support for a serial-founder migration model. We further show that Bantu speakers received significant gene flow from local groups in regions they expanded into. Our genetic dataset provides an exhaustive modern-day African comparative dataset for ancient DNA studies 10 and will be important to a wide range of disciplines from science and humanities, as well as to the medical sector studying human genetic variation and health in African and African-descendant populations.