Samples of skin tissue were collected by biopsy darting from humpback whales (Megaptera novaeangliae) in six seasonal habitats representing three stocks and four regions: Groups IV (western Australia), V western component (eastern Australia), V eastern component (New Zealand and Tonga) and VI (the Antarctic Peninsula and Gorgona Island, Colombia, South America) of the Southern Hemisphere. A variable section of the mitochondrial DNA control region was amplified and sequenced from 84 of these individuals, distinguishing a total of 48 unique sequences (i. e., mtDNA nucleotypes). Phylogenetic reconstructions suggested that these nucleotypes form three clades, corresponding to those previously described in a world‐wide survey of humpback whale mtDNA variation, although bootstrap support for two of the clades was relatively low (<50%). An analysis of variance adapted for molecular information showed significant differentiation of nucleotypes among the three Groups (Stocks) and heterogeneity of haplotype diversity among the four regions. A pattern of interchange within and between oceanic basins was demonstrated by the presence of shared identical nucleotypes among humpback whales in regions of the Southern and Northern Hemispheres.