Introgressive hybridization has an important evolutionary significance in terms of gene diversity and speciation. Among the major groups of vertebrates, fish show a strong propensity to hybridize. In order to highlight the possible occurrence of gene flow between two tilapia species, Oreochromis niloticus and O. aureus, a comparison of allozyme and mitochondrial DNA (mtDNA) polymorphism was performed on sympatric and allopatric populations of these two species. Nuclear data were congruent with the morphological identification of O. niloticus and O. aureus populations. In opposition, the mtDNA analysis resulted in two strictly differentiated groups which did not follow the morphological and nuclear DNA classification. The first group consisted of East African O. niloticus populations and the second included all the O. aureus populations and the West African O. niloticus populations. Moreover, in some cases, the same sequences were detected in both species. These data strongly support a differential introgression of mtDNA from O. aureus to O. niloticus involving all the West African area. This work points out the risk of misinterpretation of mtDNA or nuclear DNA data when only one single class of marker is used.