Two mitochondrial DNA segments of the bigeye tuna (Thunnus obesus) were amplified by polymerase chain reaction (PCR), and restriction fragment length polymorphism (RFLP) analyses of these segments were used for the genetic stock study. The variation in a segment flanking the ATPase and COIII genes was low; only two genotypes (α and β) were detected by RsaI digestion. Yet a large difference in the genotype distribution was observed between ocean basin samples. The α type predominated in four Atlantic samples, where 178 of 244 individuals were the α type. In contrast, only one of 195 individuals collected in the Indo-Pacific was the α type? The frequency of the α type varied considerably from 0 to 80% among seven samples collected off the Cape of Good Hope. The variation found in the other segment, containing the D-loop region, was much higher; two endonucleases (DpnII and RsaI) detected five genotypes each and 15 composite genotypes. A highly significant difference in genotype frequencies was observed between the Atlantic and Indo-Pacific samples, but no heterogeneity was observed among the four Atlantic or among four Indo-Pacific samples. These results clearly indicate that not only gene flow, but also fish migration, between the Atlantic and Indian Oceans are severely restricted, and that fishes from these distinct stocks are intermingling around South Africa. The simple and diagnostic genetic marker found in this study can be used to estimate mixing ratios between Atlantic and Indian stocks around South Africa.