We analysed the genetic differentiation among 17 natural populations of the Nile tilapia Oreochromis niloticus (Linnaeus, 1758) using allozymes and restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA). The populations studied, from the River Senegal to Lake Tana and from Lake Manzalla to Lake Baringo, represent all subspecies which have been previously described. Sixteen variable nuclear loci showed that these populations can be clustered in three groups: (1) West African populations (Senegal, Niger, Volta and Chad drainages), (2) Ethiopian Rift Valley populations (Lakes Awasa, Ziway, Koka and the Awash River) and (3) Nile drainage (Manzalla, Cairo, Lake Edward) and Kenyan Rift Valley populations (Lakes Turkana, Baringo and River Suguta). Nine different mtDNA haplotypes were found in the RFLP analysis of a 1 kb portion of the D-loop region. The network obtained showed that there are three geographically distinct groups; all West African populations and O. aureus are clustered, the two Ethiopian Rift Valley populations are distinct and between these two groups are the Kenyan and Ugandan Rift Valley populations. Nile populations show affinities both with West African populations and with specimens from Lakes Tana and Turkana. Taxonomic and biogeographical implications of these results are discussed.