Summary: The genetic mechanisms of reproductive isolation have been widely investigated within Asian cultivated rice (Oryza sativa); however, relevant genes between diverged species have been in sighted rather less. Herein, a gene showing selfish behavior was discovered in hybrids between the distantly related rice species Oryza longistaminata and O. sativa. The selfish allele S13l in the S13 locus impaired male fertility, discriminately eliminating pollens containing the allele S13s from O. sativa in heterozygotes (S13s/S13l). Genetic analysis revealed that a gene encoding a chromatin-remodeling factor (CHR) is involved in this phenomenon and a variety of O. sativa owns the truncated gene OsCHR745, whereas its homologue OlCHR has a complete structure in O. longistaminata. CRISPR-Cas9-mediated loss of function mutants restored fertility in hybrids. African cultivated rice, which naturally lacks the OlCHR homologue, is compatible with both S13s and S13l carriers. These results suggest that OlCHR is a Killer gene, which leads to reproductive isolation.