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Hybrid sterility is a reproductive barrier that prevents gene flow between species. In Oryza species, some hybrid sterility loci, which are classified as gamete eliminators, cause pollen and seed sterility and sex-independent transmission ratio distortion (siTRD) in hybrids. However, the molecular basis of siTRD has not been fully characterized because of lacking information on causative genes. Here, we analyze one of the hybrid sterility loci, S2, which was reported more than forty years ago but has not been located on rice chromosomes. Hybrids between African rice (Oryza glaberrima) and a near-isogenic line that possesses introgressed chromosomal segments from Asian rice (Oryza sativa) showed sterility and siTRD, which confirms the presence of the S2 locus. Genome-wide SNP marker survey revealed that the near-isogenic line has an introgression on chromosome 4. Further substitution mapping located the S2 locus between 22.60 Mb and 23.54 Mb on this chromosome. Significant TRD in this chromosomal region was also observed in a calli population derived from cultured anther in hybrids of another cross combination of African and Asian rice species. This indicates that the pollen abortion caused by the S2 locus occurs before callus induction in anther culture. It also suggests the wide existence of the S2-mediated siTRD in this interspecific cross combination. Chromosomal location of the S2 locus will be valuable for identifying causative genes and for understanding of the molecular basis of siTRD.