Astragalus sinicus L. is one of cross-pollinated plants with heterogeneity in cultivar population which results in difficulty for cultivar identification. In this study, 3 cultivars selected from Minzi cultivar series bred in Fujian have been analyzed by using SSR molecular marker and the effect of sampling strategy on cultivar differentiation was investigated. The results showed that: (1) When the primer combination was confirmed by 4 pairs and the sampling amount ranged from 5 to 50 in each cultivar, the amplified loci and the observed number of alleles tended to increase in the cultivar, but the effective number of alleles，Shannon index，and genetic diversity index tended to decline after reaching to a maximum as sampling amount was as much as 30 for total sample; With the increase of the amount of sample, the Nei’s genetic distance between cultivars and the PhiPT value among population obtained from AMOVA reduced，but the confidence level of PhiPT was raised. (2) When the sample capacity was constant with 30 or 50, and the primer number was increased to 6 with the added 2 primers amplifing the marking locus of different frequency among the cultivars, the tested cultivars could be effectively identified because of the higher PhiPT value and more significant confidence level (P<0.001). The analysis of principal component further indicated that the distribution of samples from 3 tested cultivars along principal component axis was similar between sampling amount of 30 and 50 . This study of sampling strategy for Astragalus sinicus indicated that: In order to enhance the ability in identification of cultivar of Astragalus sinicus L., the optimum sampling amount was as much as 30 that could achieve the better effect for cultivar identification as well as reduce the cost in analysis.