Technical Abstract: Alfalfa (Medicago sativa L.) is one of the most important cultivated forage legumes worldwide. Understanding the areas of adaptation and genetic variation available in a crop species facilitates efforts to identify suitable germplasm for integration in plant breeding programs. Accessions that represent the USDA perennial Medicago collection includes M. sativa subsp. sativa, M. sativa subsp. varia, M. sativa subsp. falcata and M. sativa subsp. caerulea originating from multiple countries were evaluated under field conditions and assayed for their genetic variation using molecular markers. The experimental design was a randomized complete block design with four replications. Data from all accessions for overall performance, plant survival, flower color, pod production, fall dormancy and persistence were collected. Aboveground biomass was harvested and relative water content (RWC) from selected accessions that represent the range of overall performance in the entire collection was measured after growth under drought conditions. Significant variation for all traits evaluated was observed among the accessions. These findings will contribute to the efficient use and conservation of genetic resources in alfalfa and enable identification of accessions suitable for unique target environments focused on specific breeding objectives.