Oryzias latipes and Oryzias curvinotus are closely related medaka species that have the common sex-determining gene, DMY, on their homologous Y chromosomes. We previously reported that sex-reversed XY females were produced in hybrids between O. curvinotus females and O. latipes males (Hd-rR inbred strain). In this study we used HNI inbred strain males of O. latipes for mating with O. curvinotus females, and found that all the XY hybrids developed as males. To map the factor responsible for this strain-specific XY sex reversal, O. curvinotus females were mated with two Y-congenic strains (HNI.Y(Hd-rR) and Hd-rR.Y(HNI)) and a recombinant congenic strain (Hd-rR.Y(HNI)rr). HNI.Y(Hd-rR) produced sex-reversed females in the XY hybrids, whereas no sex-reversed females were obtained in the XY hybrids from Hd-rR.Y(HNI) and Hd-rR.Y(HNI)rr, demonstrating that a small region on the Y chromosome, which includes DMY, is responsible for the XY sex reversal. Sex-reversed hybrids were only produced in the presence of the Y-chromosomal region derived from the Hd-rR strain, suggesting that missense or regulatory mutations specific to the Hd-rR Y-chromosomal region induce the sex reversal.