To assess the indica-japonica differentiation of improved rice varieties, a total of 512 modern varieties including 301 indica and 211 japonica accessions were analyzed using 36 microsatellites. The Fst coefficients ranged from 0.002 to 0.730 among the loci with an average of 0.315. Significant differentiation was detected at 94.4% of the loci studied (P<0.05, pairwise Fst tests), indicating that there was a high level of indica-japonica differentiation within the improved varieties. At 18 loci, about 74%-98% of the alleles of indica and japonica accessions were distributed in two ranges of amplicon length. Linkage disequilibrium analysis showed that the distribution trends were significantly nonrandomly associated. Using the differentiation trends at the 18 loci, microsatellite index (MI) was proposed for discrimination of the two subspecies. When rice accessions with MI value greater than zero were classified as indica, and those with MI value smaller than zero were classified as japonica, about 96.1% of the accessions could be classified. This result agrees with the classification based on morphological-physiological characters, indicating that this method is feasible and effective. To assess the indica-japonica differentiation of improved rice varieties, a total of 512 modern varieties including 301 indica and 211 japonica accessions were analyzed using 36 microsatellites. The Fst coefficients ranged from 0.002 to 0.730 among the loci with an average of 0.315. Significant differentiation was detected at 94.4% of the loci studied (P <0.05, pairwise Fst tests), indicating that there was a high level of indica-japonica differentiation within the improved varieties. At 18 loci, about 74% -98% of the alleles of indica and japonica accessions were distributed in two ranges of amplicon length. Linkage disequilibrium analysis showed that the distribution trends were significantly nonrandomly associated. Using the differentiation trends at the 18 loci, microsatellite index (MI) was proposed for discrimination of the two subspecies. rice accessions with MI value greater than zero were classified as indica, and those with MI value smaller than zero were classified as jap onica, about 96.1% of the access could could be classified. This result agrees with the classification based on morphological-physiological characters, indicating that this method is feasible and effective.