Abstract: Herbaceous peony(Paeonia lactiflora Pall.)is a perennial herbaceous plant which belongs to Sect. Paeonia in family Paeoniaceae. It is a traditional flower with 3000-year history and has hundreds of cultivars in China. However, it is difficult to distinguish cultivars accurately only by the traditional morphological identification method. Establishing the multi-level molecular identification techniques can improve the accuracy of cultivar identification. In order to develop a molecular identification method,we used the SSR technique to establish the molecular ID code of 66 cultivars of Chinese traditional herbaceous peony. In this research, 111 pairs of SSR primers with 84 from literatures and 27 self-designed, were labeled with 6% none-denaturing polyacrylamide gel electrophoresis and quadruple fluorescence capillary electrophoresis. Then 29 pairs of primers with high polymorphism and good repeatability were selected. The results showed that 223 polymorphic alleles were revealed by the 29 primer pairs,with an average of 7.69 alleles per pair. The effective number of alleles (Ne) was 3.658±0.328, Shannon diversity index (I) was 1.371±0.104, observed heterozygosity (Ho) was 0.532±0.045, expected heterozygosity (He) was 0.645±0.036, and the range of PIC at 29 SSR loci of 66 cultivars was 0.20-0.84, 0.60 on average. Based on the principle of distinguishing the most varieties using the minimum pairs of primers, 4 pairs of primers (Pmg153, Knu73, Pmg209, J38) were selected out. This study indicates that the 4 pairs of primers can identify the 66 varieties of Chinese traditional herbaceous peony and can successfully establish 66 molecular ID codes by different single digits and uppercase letters. It not only provides a rapid, accurate and efficient molecular identification method for herbaceous peony cultivars, but also contributes to genetic improvement of cultivars of Chinese traditional herbaceous peony and is significant for protection of the new varieties.