LUCERNE, alfalfa (Medicago sativa L). is a major forage crop of family Fabaceae. The present study was concerned with the genetic discrimination and identification of some M. sativa cultivars. Nine M. sativa cultivars, representing three different countries (Egypt, Australia and USA), were presented in this study. The genetic relatedness was studied using DNA barcoding of rbcL and rpoC1 marker genes; Phylogenetic analysis of samples of nine M. sativa cultivars was performed and a cluster analysis was derived. Sequence analysis was in complement and efficient in assessing the characterization of genetic relatedness between the studied M. sativa cultivars and has revealed that the Egyptian cultivars are more phylogenetically related to the American cultivars (USA 9: Perfect and USA4: Cuf101) and the Australian one (AUS 2: Sirinafa). In conclusion, the results have indicated that DNA barcoding was suitable and crucial for characterization of M. sativa at species and cultivar level, which provides an important tool for future analyses of other major important forage crops. Moreover, it might be suggested that breeding lines from M. sativa cultivars may develop novel insights and give better understanding of the possibilities of domestication of M. sativa. On the same context, some cultivars could be used as important genetic resources for genetic improvement of M. sativa in future breeding programs.