Abstract: The genetic structure and diversity of 25 alfalfa (Medicago sativa) salt-tolerant varieties was analyzed by RAPD technology. Results showed that the polymorphism loci percentages (P) of 30 primers between single-plant DNA of 25 salt-tolerant alfalfa varieties were 81.52%, and between mixed DNA of various varieties were 61.65%, indicating that single-plant DNA sample was a better method than mixed DNA sample, to reveal the level of genetic variation within and between alfalfa varieties. The coefficient of gene differentiation (Gst) mainly reflects the ratio of species variation to total variation, and the Gst of 18 Chinese salt-tolerant alfalfa varieties and 7 American salt-tolerant alfalfa varieties were 0.271 and 0.152, respectively, showing that there was more interactive gene exchange between Chinese salt-tolerant alfalfa germplasm was than that of American germplasm. Alfalfa is a typical outcrossing plant, and there was a direct link between their genetic structure and breeding system. Based on genetic distance (GD) analysis, 9 groups were obtained from the 25 varieties, of which, the GD of No.1 and No.2 Tumu was the least (GD=0.148), and that of No.1 Jieda and Tumu was the greatest (GD=0.786). Genetic diversity analysis of salt-tolerant alfalfa germplasm provided a theoretical basis for the repository building of alfalfa salt-tolerant core germplasm and for the selection and breeding of new salt-tolerant varieties.