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A sharp genetic break separates Atlantic from Indo-Pacific bigeye tuna (Thunnus obesus) populations, as the frequencies of two major mitochondrial (mt) DNA types ([alpha] and [beta]) found in this species are different across the tip of southern Africa. The level of nucleotide divergence between mtDNA types [alpha] and [beta] is of the same order as that between reproductively isolated taxa. To further investigate the genetic structure of bigeye tuna over its distribution range and in the contact zone off southern Africa, bigeye tuna samples collected between 1992 and 2001 (including samples from a previous mtDNA survey) were characterized for four nuclear DNA loci and for mtDNA. Nuclear markers did not support the hypothesis that [alpha] and [beta] mitochondria characterize sibling species. Significant allele-frequency differences at one intronic locus (GH2) and one microsatellite locus ([micro]208) were found between Atlantic and Indo-Pacific samples, although the level of nuclear genetic differentiation (Weir and Cockerham's [theta] = 0.025 to 0.042) was much lower than in mtDNA ([theta] = 0.664 to 0.807). Probabilistic Bayesian assignment of individuals to a population confirmed that southern African bigeye tuna samples represent a simple mixture of individuals from Atlantic and Indian stocks that do not interbreed, with a higher contribution from Indian Ocean individuals (about 2/3 vs. 1/3).