To refine our understanding of the spatial structure of the green turtle (Chelonia mydas) populations in the South West Indian Ocean (SWIO), we analysed patterns of mitochondrial DNA (396 base pairs control region fragment) variation among 171 samples collected at five distinct locations (Kenya, Northern Mozambique, and three locations in the Republic of Seychelles: the Granitic, Amirantes, and Farquhar groups) and compared them to genetic data (n = 288), previously collected from 10 southern locations in the SWIO. We also analysed postnesting satellite tracks (n = 4) from green turtles nesting in the Amirantes group. Pairwise comparisons of haplotype frequencies showed significant genetic differentiation amongst rookeries and suggest that the SWIO hosts two main genetic stocks of nesting green turtles that could themselves be divided in two sub-stocks that still need to be confirmed: A. the Southern Mozambique Channel, that could be composed of two sub-stocks (a1) Europa and (a2) Juan de Nova, and B. the Northern SWIO (N-SWIO) comprising two sub-stocks (b1) the Seychelles archipelago stock—SEY; and (b2) the remaining Northern SWIO rookeries. The newly revealed differentiation of the Seychelles population is supported by restricted migration of females tracked from the Amirantes group suggesting relatively limited links with other regional stocks. We hypothesize that this differentiation could be due to local and regional current patterns and to the role of the Indo-Pacific Barrier as a genetic break, enhanced during periods of sea level decrease associated with a rare but continuous flow of hatchlings and young juveniles from Western Australia.