The genetic structure of seven sailfish Istiophorus platypterus populations sampled from three locations inside and four locations outside the Arabian Gulf was determined by restriction fragment length polymorphism analysis of mitochondrial DNA of 147 individuals using eight restriction endonucleases. A total of 39 composite haplotypes derived from 27 presumptive restriction sites demonstrated significant differences in frequency between population groups inside and outside the Gulf (analysis of molecular variance 34.80%, P<0.001; F-ST=0.356) and evidence of restricted migration between them (average number of migrants, N-m=0.903). Haplotypes found only inside or outside the Gulf clustered to all major branches of a haplotype phylogeny, as did those found in both areas. The reduced genetic diversity of the Gulf populations and the fact that much of the differentiation between the population groups resulted from differences in haplotype frequency rather than divergence between haplotypes suggest a founder effect and a recent sampling of genotypes from the Indian Ocean. This was probably associated with dispersal into the Gulf after it was flooded by rising sea level after the end of the last glaciation around 8,000 years ago. At some point since then the population has evolved to complete its life cycle within the Gulf and shows a marked disruption to gene flow, consistent with dispersal data, at the Strait of Hormuz. These findings represent the first clear evidence of phylogeographic isolation occurring in a large, highly vagile, predatory istiophorid billfish, within a marginal sea.