Abstract：EST-SSR marker is a new kind of SSR markers which is developed from the ESTs（expressed sequence tags）. This novel type molecular maker was part of expressed gene, therefore, when used in Brassica napus genetic analysis, they could reveal differences in related gene expression among varieties. In this study, a total of 21 EST-SSR markers were used to analyze the genetic diversity of 42 Brassica napus varieties. All the primer sets produced clear and strong amplifications, and 85 alleles were generated and examined. 49 alleles of all showed polymorphic among the 42 Brassica napus varieties, accounting for 57. 65%. The range of the 42 varieties’ genetic distance was 0.0087-0.1885 computed by NTSYS（software）. when GD on 0.1508, the varieties were divided into 5 groups, which indicated difference of these germplasm based on different cultural area approximately.
