Abstract: In this study, we analyzed 84 SSR primer pairs from closely related species and designed 23 EST-SSR primer pairs from NCBI for developing more mei flower(Prunus mume Sieb. et Zucc.)new SSR makers. Twelve mei flower cultivars were used to do polymorphic test on these primers. The results show that 69 SSR primer pairs can amplify bands with the efficient amplification ratio of 82.1%. A total of 85 alleles were detected by 14 primer pairs with an average of six effective alleles per primer. The effective number of alleles was between 2.072 and 5.539, with the average of 4. The polymorphic information content (PIC) value was between 0.483 and 0.786, with the average of 0.695. The average Shannon Index was 1.528, with the genetic similarity coefficient among 12 materials ranging from 0.13 to 0.60 and the average of 0.26. Based on the UPGMA analysis, P. mume ‘Yudie Longyou’ and P. mume ‘Xiao Yudie’ contained similar inheriting basement, about 0.60, and the similarity value between P. mume ‘Meiren’ and P. mume ‘Huangjin’ was smallest, about 0.13. The designed 22 EST-SSR primer pairs can amplify bands, with the effective amplificaition rate of 95.6%. Eleven primer pairs demonstrated the polymorphic characteristics, accounting for 47.8% of all the EST-SSR.