Epistasis is considered to be a primary genetic basis of hybrid breakdown. We found novel epistatic genes causing hybrid breakdown in an intraspecific cross of cultivated rice (Oryza sativa L.). F2 progeny derived from a cross between a Japonica variety, Asominori, and an Indica variety, IR24, showed segregation of high sterility for seeds, even though the reciprocal F1 hybrids showed about 60% seed fertility. Backcross populations (BC3F2, BC3F3), obtained from repeated backcrossing with Asominori, showed the segregation of causal genes in a simple Mendelian fashion. Using these populations, we identified that this sterility was hybrid breakdown caused by interaction among three nuclear genes distributed on the both parental genomes. These new genes, designated as hsa1, hsa2, and hsa3, were found to be involved in female gamete development by histological examination. The Indica parent IR24 has a sterile allele, hsa1-IR, which was located at near RFLP marker G148 on chromosome 12, whereas the Japonica parent Asominori has two sterile alleles, hsa2-As on chromosome 8 (close to G104) and hsa3-As on chromosome 9 (close to RM285). Female gametes carrying the hsa1-IR, hsa2-As, and hsa3-As alleles aborted in hsa1-IR homozygous plant, leading to seed sterility and selective elimination of the specific allelic combination. This study provides direct evidence that hybrid breakdown is attributed to epistatic interaction of genes from both parents and suggests that complicated mechanisms has been developed for hybrid breakdown during the evolution of rice.