This study addresses the challenge of arid conditions in drylands by focusing on fortifying the resilience of alfalfa within an ongoing varietal improvement initiative. The assessment encompasses five pre-selected alfalfa (Medicago sativa L.) populations bred in The Institute of Arid Regions, Medenine (Tunisia). The primary objective is to formulate an alfalfa genetic pool that is optimized for breeders and farmers in arid regions and emphasizes chemical composition, biochemistry, in vitro digestibility (IVDMD), and relative feed value (RFV). Significant differences (p < 0.05) among alfalfa populations were observed for all parameters except acid detergent fiber (ADF) and IVDMD. Key variations include crude protein (CP) (15.21-21.82%), neutral detergent fiber (NDF) (42.96-53.78%), ADF (31.13-39.17%), RFV (111.96-122.73), IVDMD (55.14-61.41%), ash (9.74-12.45%), total polyphenols (29.95-39.55 mg GAE g-1 DM), and total flavonoids (26.4-35.15 mg QE g-1 DM). Crucially, multivariate analysis identified superior performers, specifically the populations "AME" and "GAB." These findings will guide ongoing genetic enhancement initiatives and contribute to the development of an alfalfa variety that is explicitly tailored to arid agricultural settings, thereby advancing sustainable agriculture in challenging environments.