It is generally accepted that Oryza rufipogon is the progenitor of Asian cultivated rice (O. sativa). However, how the two subspecies of O. sativa (indica and japonica) were domesticated has long been debated. To investigate the genetic differentiation in O. rufipogon in relation to the domestication of O. sativa, we developed 57 subspecies-specific intron length polymorphism (SSILP) markers by com- parison between 10 indica cultivars and 10 japonica cultivars and defined a standard indica rice and a standard japonica rice based on these SSILP markers. Using these SSILP markers to genotype 73 O. rufipogon accessions, we found that the indica alleles and japonica alleles of the SSILP markers were predominant in the O. rufipogon accessions, suggesting that SSILPs were highly conserved during the evolution of O. sativa. Cluster analysis based on these markers yielded a dendrogram consisting of two distinct groups: one group (Group I) comprises all the O. rufipogon accesions from tropical (South and Southeast) Asia as well as the standard indica rice; the other group (Group II) comprises all the O. rufipogon accessions from Southern China as well as the standard japonica rice. Further analysis showed that the two groups have significantly higher frequencies of indica alleles and japonica alleles, respectively. These results support the hypothesis that indica rice and japonica rice were domesticated from the O. rufipogon of tropical Asia and from that of Southern China, respectively, and suggest that the indica-japonica differentiation should have formed in O. rufipogon long before the beginning of domes-tication. Furthermore, with an O. glaberrima accession as an outgroup, it is suggested that the indica-japonica differentiation in O. rufi-pogon might occur after its speciation from other AA-genome species. However, the two subspecies of O. sativa (indica and japonica) were domesticated has long been debated. To investigate the genetic differentiation in O. It is generally accepted that Oryza rufipogon is the progenitor of Asian cultivated rice (O. sativa). rufipogon in relation to the domestication of O. sativa, we developed 57 subspecies-specific intron length polymorphism (SSILP) markers by com- parison between 10 indica cultivars and 10 japonica cultivars and defined a standard indica rice and a standard japonica rice based on these SSILP markers. Using these SSILP markers to genotype 73 O. rufipogon accessions, we found that indica alleles and japonica alleles of the SSILP markers were predominant in the O. rufipogon accessions, suggesting that SSILPs were highly conserved during the evolution of O. sativa . Cluster analysis based on these markers yielded a dendrogram consisting of two distinct groups: one group (Group I) all all the O. rufipogon accesions from tropical The South and Southeast Asia as well as the standard indica rice; the other group (Group II) includes all the O. rufipogon accessions from Southern China as well as the standard japonica rice. Further analysis showed that the two groups have of indica alleles and japonica alleles, respectively. These results support the hypothesis that indica rice and japonica rice were domesticated from the O. rufipogon of tropical Asia and from that of Southern China, respectively, and suggest that the indica-japonica differentiation should have formed in O. rufipogon long before the beginning of domes-tication. Furthermore, with an O. glaberrima accession as an outgroup, it is suggested that the indica-japonica differentiation in O. rufi-pogon might occur after its speciation from other AA-genome species.