The embryo-sac is an essential structure for angiosperm reproduction. The cytological and genetic characterization of embryo-sac sterility was examined in a cross between Oryza sativa ssp. indica cv. ZYQ8 and ssp. japonica cultivar, JX17. The arrest of embryo-sac development was manifested following meiosis in the F1 hybrid. When the megaspore carried the lethal genotype, the nucleus either failed to divide or divided only once, and the immature embryo-sac degenerated. Abortion of the embryo-sac in the indica-japonica hybrid background was not observed in their original parents, and an effect of cytoplasmic gene(s) on embryo-sac sterility in the reciprocal F1 hybrids was not detected. Using a rice molecular linkage map based on a doubled haploid (DH) population from the cross of ZYQ8 /JX17, we mapped quantitative trait loci (QTLs) for the defective development of the female gametophyte in backcross progenies from the DH lines. The result demonstrated that a polygenic system is involved in both megagametogenesis and postzygotic isolation in inter-subspecific hybrid rice.