Sex determination (SD) in Nile tilapia involves a major genetic factor following an XX/XY pattern but can also be influenced by minor genetic factors and temperature. So far, studies on sex determination in this species have been carried out on commercial/laboratory strains. However, the linkage group carrying the major sex-determinant varies between the strains studied and associations with LG1, LG23 or both have been proposed. The SD on LG1 remains to be discovered. On LG23, a tandem duplication of the amh gene seems to be responsible for maleness. By studying three natural populations (one of O. niloticus niloticus originating from Ghana and two of O. niloticus cancelatus from Ethiopia), and by coupling long and short reads from whole genome sequencing, we were able to show that the SD region is polymorphic in the wild. The system previously described in LG23 laboratory strains was found in the Ghanaian and in one of the Ethiopian populations. We described the structure of the Y haplotype with precision specifying the breakpoints of the duplication, larger than the amh gene along with population specific polymorphism. However, no association to sex was seen in the other Ethiopian population on LG23 and no amh duplication was found in males. Although the Ethiopian populations are geographically and phylogenetically closer to each other, it appears that the ancestral sex-determination region may be lost and replaced potentially by a polygenic system or by an environmental SD.