Hybrid sterility is a major form of postzygotic reproductive isolation. Although reproductive isolation has been a key issue in evolutionary biology for many decades in a wide range of organisms, only very recently a few genes for reproductive isolation were identified. The Asian cultivated rice (Oryza sativa L.) is divided into two subspecies, indica and japonica. Hybrids between indica and japonica varieties are usually highly sterile. A special group of rice germplasm, referred to as wide-compatibility varieties, is able to produce highly fertile hybrids when crossed to both indica and japonica. In this study, we cloned S5, a major locus for indica-japonica hybrid sterility and wide compatibility, using a map-based cloning approach. We show that S5 encodes an aspartic protease conditioning embryo-sac fertility. The indica (S5-i) and japonica (S5-j) alleles differ by two nucleotides. The wide compatibility gene (S5-n) has a large deletion in the N terminus of the predicted S5 protein, causing subcellular mislocalization of the protein, and thus is presumably nonfunctional. This triallelic system has a profound implication in the evolution and artificial breeding of cultivated rice. Genetic differentiation between indica and japonica would have been enforced because of the reproductive barrier caused by S5-i and S5-j, and species coherence would have been maintained by gene flow enabled by the wide compatibility gene.