Abstract: Genomic DNA was extracted by advanced CTAB methods from Medicago sativa. The L₁₆(4⁵) orthogonal diagram was applied to study the effects of mobility reproducible in different concentrations of template DNA, dNTPs, Mg²⁺, Taq polymerase, and primer on M. sativa. A suitable SRAP reaction system had 50 ng DNA,1.4 mmol/L Mg²⁺,1.2 U Taq polymerase, 0.4 mmol/L dNTPs and 0.25 μmol/L in a 20 μL reaction system. Better primers were screened out from 100 pairs of primers using optimizated systems, that could amplify clear fragments. A total of 87 amplicons were resolved by 8 pairs of well-chosen primers, of which 84 were polymorphic. These varieties had high genetic polymorphisms. A dendrogram from the analysis clustered two Medicago hispida cultivars from Anhui and Jiangsu into one group. Thirty two other cultivars were classified into sub-groups with a similarity coefficient of 0.68. Fall dormancy alfalfa cultivars were mostly distributed in the second sub-group. Semi-fall dormancy and non-fall dormancy alfalfa cultivars did not fall in the same sub-group. There was no absolute distinction of fall dormancy of alfalfa.