The genetic relationships of 42 alfalfa varieties were studied using RAPD analysis. The results showedthat it is helpful to improve the genomic DNA quality used for RAPD amplification by using modified CTAB meth-od. 19 random primers composed of 10 bases were selected from the RAPD markers, and there were 138 polymor-phism fragments in the 220 amplified fragment found, accounting for 62.7%, 82 monomorphism fragments, account-ing for 37.3%. MEGA2.1 software was used for calculating genetic distance of 42 alfalfa varieties. There were a dis-tant relationship among them because the range of genetic distance was 0.174-0.5000. 42 alfalfa varieties can be di-vided into 5 groups when genetic distance was 0.435. RAPD markers can be used as the basis for classification of al-falfa genetic relationship, because the genetic relationship of different alfalfa varieties have bigger difference. Thestudy was done to provide reliable scientific basis for improvement and hybrid parents selection of alfalfa variety.