The sex-determining gene in Oryzias latipes and O. curvinotus has been proved to be DMY. Although O. curvinotus has the DMY gene on the Y chromosome which is homologous to the Y chromosome of O. latipes, the sex-determining mechanism of other Oryzias fishes has not been identified. In order to uncover the sex-determining mechanism of O. luzonensis and O. mekongensis, which are most closely related species to O. latipes and O. curvinotus, we analyzed the sex ratio of the progeny of sex-reversed fish. We were able to obtain sex-reversed males by the administration of methyltestosterone, and found that these yielded all-female offspring in both species. These results indicate that O. luzonensis and O. mekongensis have the XX-XY sex-determination system.