Abstract: Systematic characterization of genetic and molecular mechanisms in the formation of hybrid sterility is of fundamental importance in understanding reproductive isolation and speciation. Using ultra-high-density genetic maps, 43 single-locus quantitative trait loci (QTLs) and 223 digenic interactions for embryo-sac, pollen, and spikelet fertility are depicted from three crosses between representative varieties of japonica and two varietal groups of indica, which provide an extensive archive for investigating the genetic basis of reproductive isolation in rice. Ten newly detected single-locus QTLs for inter- and intra-subspecific fertility are identified. Three loci for embryo-sac fertility are detected in both NipxZS97 and NipxMH63 crosses, whereas QTLs for pollen fertility are not in common between the two crosses thus leading to fertility variation. Five loci responsible for fertility and segregation distortion are observed in the ZS97xMH63 cross. The importance of two-locus interactions on fertility are quantified in the whole genome, which identify that three types of interaction contribute to fertility reduction in the hybrid. These results construct the genetic architecture with respect to various forms of reproductive barriers in rice, which have significant implications in utilization of inter-subspecific heterosis along with improvement in the fertility of indica-indica hybrids at single- and multi-locus level.