To evaluate the application of SSR markers in pepper DUS testing, here we employed thirty pairs of SSR primers in fingerprinting analysis of 130 varieties. A total of 163 alleles were detected in 130 accessions, which ranged from 2 to 15 with a mean value of 5.4. The polymorphism information content (PIC) ranged from 0.17 to 0.76, with the average of 0.43. These genotypes can be clarified using the polymorphic information. These pepper cultivars with close genetic background were clustered. The genetic distance of the 130 cultivars ranged from 0.01 to 0.91.There were 20 pairs of cultivars whose genetic distance was less than 0.10, among which 3 pairs had one locus difference. It was more suitable to judge the distinctness of pepper varieties by using the number of difference locus. Ten pairs of primers were used to detect the uniformity of the variety 0313963-4 and the comprehensive uniformity ratio R value was 96.5, which was lower than the requirement of phenotypic trait in DUS test. Therefore, SSR markers directly used in pepper DUS determination still need further study, but it can be used in the selection of Similar varieties.