Cichlid species of the genus Oreochromis vary in their genetic sex-determination systems. In this study, we used microsatellite DNA markers to characterize the sex-determination system in Oreochromis tanganicae. Markers on linkage group 3 were associated with phenotypic sex, with an inheritance pattern typical of a female heterogametic species (WZ-ZZ). Further, locus duplication was observed for two separate microsatellite markers on the sex chromosome. These results further advance our understanding of the rapidly evolving sex-determination systems among these closely related tilapia species.