Two test cross populations were developed by crossing a set of DH lines as male parents to two wide compatibility rice lines, photoperiod-sensitive genic male sterile (PGMS) line-N422S and thermosensitive-genic male sterile (TGMS) line-Peiai64S. Polymorphism of the cross parents and another set of diverse indica or japonica lines (as a control) was assayed by using 92 RFLP markers. 41 RFLP markers were detected highly associated with indica and japonica phenotypes, which can be used as diagnostic markers to differentiate indica and japonica. Our results indicated that 87.8% of the diagnostic markers were also highly associated with grain yield and its components in at least one of the test cross populations, suggesting parallel relationships between the genes involving in evolution and QTLs controlling grain yield and yield components in the process of differentiation of rice (O. sativa L.). Further analysis indicated that fertility was a main factor affecting the heterosis for grain yield in inter-subspecific rice hybrids. The fertility was conditioned by both intra-locus and inter-locus gene interactions and favorable genic interactions could raise it accordingly.