Abstract:Analysis of molecular genetic diversity in Alfalfa(Medicago sativa L.) germplasm is of great importance for conserving and breeding programs. The objective of this study was to evaluate the genetic diversity of alfalfa varieties, and to identify them through screening the variety-specific primers. This report analyzed the genetic diversity of 16 alfalfa varieties from Gansu Province based on RAPD markers using bulking equal quantities of DNA from 20 individuals of a cultivar. Based on the Roger’s distances between the sixteen cultivars, the dendrogram by UPGMA cluster analysis showed that the relationships among these cultivars were tightly related to breeding programs. There was a clear difference between creeping variety Jindera and other erect varieties. Within the erect accessions, 3 distinct groups including one introduced varieties and synthetic varieties originated from alien germplasm, one east-central group and one west group, were detected. Four of 10 primers, OPE4, OPE5, OPE6 and OPE7, amplified the specific bands belong to 5 cultivars, Gannong 3, Ganza 27, Jindera, Longdong and Algoniun, were detected, respectively, which could be used to identify alfalfa varieties through exploring the variety-specific primers.