The molecular evolution of cultivated rice Oryza sativa L. has long been a subject of rice evolutionists. To investigate genetic diversity within and differentiation between the indica and japonica subspecies, 22 accessions of indica and 35 of japonica rice were examined by five microsatellite loci from each chromosome totalling 60 loci. Mean gene diversity value in the indica rice (H=0·678) was 1·18 times larger than in the japonica rice (H=0·574). Taking the sampling effect into consideration, average allele number in the indica rice was 1·40 times higher than that in the japonica rice (14·6 vs 10·4 per variety). Chromosome-based comparisons revealed that nine chromosomes (1, 2, 3, 4, 5, 8, 9, 10 and 11) harboured higher levels of genetic diversity within the indica rice than the japonica rice. An overall estimate of FST was 0·084–0·158, indicating that the differentiation is moderate and 8·4–15·8% of the total genetic variation resided between the indica and japonica groups. Our chromosome-based comparisons further suggested that the extent of the indica–japonica differentiation varied substantially, ranging from 7·62% in chromosome 3 to 28·72% in chromosome 1. Cluster analyses found that most varieties formed merely two clusters for the indica and japonica varieties, in which two japonica varieties and five indica varieties were included in the counterpart clusters, respectively. The 12 chromosome-based trees further showed that 57 rice varieties cannot be clearly clustered together into either the indica or japonica groups, but displayed relatively different clustering patterns. The results suggest that the process of indica–japonica differentiation may have proceeded through an extensive contribution by the alleles of the majority in the rice genome.