The sex ratio of bitterling hybrids (subfamily: Acheilognathinae) is often likely to be biased toward males. Artificial hybridization was carried out in 10 species of bitterlings (three genera) in order to elucidate the masculinization mechanism of hybrids. Tanakia himantegus never produced viable F1 hybrids with other species, while hybrids of most other species were viable. In terms of sex ratio and fertility, hybrids were clearly divided into two groups: congeneric Tanakia hybrids and others. Both male and female congeneric Tanakia hybrids were fertile. The sex ratio was nearly 1:1 in all groups of Tanakia hybrids. Except for the congeneric Tanakia hybrids, sterile males appeared predominantly in groups of hybrids in which females were very rare but remained fertile. Sterile intersexes were also observed in five hybrid groups: T. lanceolata (female) x Acheilognathus cyanostigma (male), Rhodeus uyekii (female) x T. lanceolata (male), A. rhombeus (female) x T. lanceolata (male), A. rhombeus (female) x T. limbata (male), and A. tabira tabira (female) x A. cyanostigma (male). In the development of male-predominant hybrids, although hybrid and control (parental species) hatching and survival rates do not differ, no females appeared in hybrids, contrary to the controls. Taking the female heterogametic sex-determining system (ZW) and the phylogenetic relationship of bitterlings into consideration, the masculinization mechanism of hybrids in bitterlings can be explained by the interaction of two sex chromosomes, derived from each parental species. The basic genetic sex in bitterlings is male (ZZ) and the derivative is female (ZW). When parental species are related, the sex phenotype of hybrids coincides with the genetic sex. However, when the parental species differ, the sex phenotype of the ZW genotype is reversed to become male by an abnormal interaction between the Z and W chromosomes. The rare appearance of females and intersexes in male-predominant hybrids might be due to complete or partial functional expression of the W chromosome.