The aim is to explore the genetic similarity(GS) of major improved rice (Oryza Sativa L.) varieties in Yunnan province The phenotypes of 17 agronomic traits and 48 SSR markers were applied to evaluate the GS among the 40 major improved rice varieties bred by 18 different breeding groups in Yunnan province since 1960s. .The average GS coefficients were 0.244 of the total 40 varieties, 0.289 of the indica ones , 0.309 of the Japonica ones and 0.162 of between indica ones and Japonica ones,respectively on the basis of 17 agronomic traits while the average GS coefficients were 0.383, 0.318，0.478 and 0.267 correspondingly on the basis of 48 SSR markers. The GS at agronomic level was lower than that at DNA level. In total ,214 number of alleles (Na) were detected,and the mean Na was 4.458 ranging from 2 to 8. The average number of the effective alleles (Ne) was 2.8336 ranging from 1.1515 to 5.2981. The average of polymorphic information contents (PIC) was 0.6058 varying from 0.2118 to 0.8816. The average genotype diversity index (H′) was 1.1328 varying from 0.3768 to 1.8087. The markers including RM84, RM249, RM152, RM222 and RM528 were found to be more suitable to evaluate improved rice varieties in Yunnan province in genetic similarity. Clustering analysis based on GS coefficients showed that all the varieties could be grouped into two obviously different clusters corresponding to the two subspecies, indica and japonica. The results also indicated that genetic difference of among the indica rice varieties was higher than that of among the japonica ones. It was discovered that the genetic diversity of improved japonica rice varieties was lower compared with the indica ones and genetic diversity of varieties bred by one breeding group was lower than that by different groups.