The taxonomic group Medicago sativa-falcata continuum includes the important cultivated forage legume, alfalfa, along with a number of other perennial, outcrossing, and morphologically differentiated subspecies at diploid and tetraploid levels. Prior information of morphology, ploidy, and genome size is vital for accurate classification of the taxa included in the complex and thus for effective usage of genetic resources in alfalfa breeding programs. The United States Department of Agriculture-National Plant Germplasm System (USDAGRIN) has an extensive collection of the members of Medicago sativa-falcata continuum gathered from all the centres of diversity. However, accessions classified in the complex are occasionally misidentified. Furthermore, the accessions identified as M. sativa subsp. ×varia (T. Martyn) Arcang. in the USDA GRIN collections have not been evaluated based on morphological traits, ploidy level or genome size. In this study, we evaluated morphological traits and determined ploidy levels and genome size of plants from 25 wild accessions classified as Medicago sativa L. subsp. ×varia using flow cytometry. All of the accessions classified as subsp. ×varia were found to be tetraploid; however, deviations from flower colour, pod shape expectations were observed. This will be a major step toward effective utilization of germplasm resources classified as M. sativa subsp. ×varia.