In the eastern South Atlantic Ocean (Region B) humpback whales are distributed along the west coast of South Africa and winter in the Gulf of Guinea. The most recent data available suggest that Breeding stock B is possibly sub-structured, with B1 considered a breeding ground and B2 a summer feeding ground and a winter migration corridor. However questions remain over the population structure of B stock. Here, we present an assessment of temporal population structure in humpback whales on the west coast of Africa using maternally (mitochondrial DNA control region) and bi-parentally (10 microsatellites) inherited markers. We amplified, sexed, genotyped and sequenced a total of 2018 samples from B1 (Gabon, Angola, São Tomé) and B2 (West South Africa). The results showed significantly differentiation based on haplotype frequencies (F ST) and molecular distances (Φ ST) between B1 and B2; similar results were obtained with the microsatellite data, however very low gene flow was detected the two regions. For the temporal analysis, significant results were obtained only for haplotype frequency statistics (F ST), where west South Africa seasons were significantly different from seasons in Gabon. When the samples were stratified by sex, significant differentiation at the haplotype level were found for both sexes and nucleotide level only for females. The direct detection of movements by genetically identified individuals, females and males, suggests that interchange occurs between regions. However, all movements to date are from a northbound to southbound direction. The results presented here indicate that there is some spatial and temporal population substructure in humpback whales in B stock.