Although humpback whales are among the best-studied of the large whales, population boundaries in the Southern Hemisphere (SH) have remained largely untested. We assess population structure of SH humpback whales using 1,527 samples collected from whales at fourteen sampling sites within the Southwestern and Southeastern Atlantic, the Southwestern Indian Ocean, and Northern Indian Ocean (Breeding Stocks A, B, C and X, respectively). Evaluation of mtDNA population structure and migration rates was carried out under different statistical frameworks. Using all genetic evidence, the results suggest significant degrees of population structure between all ocean basins, with the Southwestern and Northern Indian Ocean most differentiated from each other. Effective migration rates were highest between the Southeastern Atlantic and the Southwestern Indian Ocean, followed by rates within the Southeastern Atlantic, and the lowest between the Southwestern and Northern Indian Ocean. At finer scales, very low gene flow was detected between the two neighbouring sub-regions in the Southeastern Atlantic, compared to high gene flow for whales within the Southwestern Indian Ocean. Our genetic results support the current management designations proposed by the International Whaling Commission of Breeding Stocks A, B, C, and X as four strongly structured populations. The population structure patterns found in this study are likely to have been influenced by a combination of long-term maternally directed fidelity of migratory destinations, along with other ecological and oceanographic features in the region. ; This material is based upon work supported by the National Research Foundation (South Africa) under Grant number 2053539. We also acknowledge Marine and Coastal Management, South Africa, for the cruises of the research vessel Algoa and collection of samples in South Africa and Mozambique, and special thanks go to Herman Oosthuizen. The Instituto Baleia Jubarte was sponsored by Petrleo Brasileiro S.A (PETROBRAS) and Norsul Cia. De Navegao. Funding to HCR was provided by numerous individuals and foundations, especially the Lenfest Oceans Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.