Abstract: Due to cross-pollinating characteristic and the excessive use of backbone materials during the alfalfa breeding, the genetic diversity of alfalfa varieties is declining, and the discrimination of different varieties through phenotypical characteristics is becoming more difficult. The construction of a DNA fingerprint database of standard alfalfa varieties through the distinctness, uniformity and stability (DUS) test can facilitate the identification of alfalfa varieties. In this study we analyzed the genetic diversity among 33 standard alfalfa varieties with a DUS test and the DNA fingerprint of each variety was constructed based on 10 SSR markers. The results showed that a total of 69 alleles were generated from the 10 SSR markers, with an average of 6.9 alleles per marker; the polymorphic ratio varied from 25% to 90%, with an average of 56.7%. The polymorphic information content per SSR marker varied from 0.56~0.87, with an average of 0.75. The 33 varieties could be clearly distinguished by the 10 SSR markers. The DNA fingerprint of each variety was established providing a technical basis for the validation and conservation of alfalfa varieties and genetic background analysis.