Previously naturally sex-reversed XY females were found in several Oreochromis niloticus populations, while it is unclear whether the naturally sex-reversed XY females could be used for breeding of YY supermale. CQ and WC O. niloticus populations were introduced. The genetic sexes of CQ and WC are controlled strictly and not strictly by LG23 chromosome, respectively. YY supermales were obtained by crossing CQ XY male fish with WC XY female fish, and whether the F₁ YY male could be used for sex control was tested. The body weight, gonadal somatic index (GSI), serum hormone level and gonadal gene expression patterns in XY and YY individuals of F₁ generation were analyzed. A quarter of YY supermales were obtained in the F₁ generation of the progeny of CQ XY male and WC XY females. F₁ YY supermales were all male and fertile. F₁ YY supermales were crossed with WC XX female, WC XY female (dam), F₁ XX female and CQ XX female, and their offspring were almost all male. Two female fish were found in the progeny of the F₁ YY male and F₁ XX female, and the male ratio is 98%. At 180 days after hatching, there were no significant differences in body weight, GSI and serum hormone levels between F₁ XY and YY hybrids. Testicular gene expression analysis showed that the expression levels of AmhX/AmhY in YY were significantly higher than those of XY fish, while the expression levels of Dmrt1 and Cyp11b2 were not significantly different between XY and YY individuals. The physiological indexes of YY fish are highly similar to those of XY fish. Naturally sex-reversed XY female O. niloticus can be used for breeding YY supermale fish, which can be used for tilapia sex control.