Alfalfa (Medicago sativa L.), the world’s most widely cultivated perennial forage crop, is valued for its high-quality feed and broad environmental adaptability. However, soil alkalization poses a major constraint to its productivity in marginal lands, with the genetic basis of alkali tolerance remaining largely unexplored. This study identified a lead SNP (Chr7-16,804,032) via a genome-wide association study (GWAS), where the T/T genotype was strongly associated with enhanced alkali tolerance. Physiological analyses revealed that lines carrying the T/T genotype maintained higher chlorophyll and soluble protein content, lower malondialdehyde levels, elevated antioxidant enzyme activities, and better Na⁺/K⁺ homeostasis under alkali stress. Functional validation across five alfalfa cultivars, supported by field trials in natural saline-alkali soils, confirmed that the T/T genotype consistently promoted biomass accumulation. Within a 14.4 Kb region flanking Chr7-16,804,032, we identified MsFAI (F-box Associated Interaction domain), a gene specifically induced under alkali stress. Overexpression of MsFAI in Arabidopsis thaliana significantly improved alkali tolerance. Further transcriptomic profiling highlighted MsCDPK (Calcium-Dependent Protein Kinase) and MsPCO (Plant Cysteine Oxidase) as alkali-responsive genes, and their heterologous expression in A. thaliana likewise conferred alkali tolerance. Taken together, our work delineates a major genetic locus (Chr7-16,804,032) and key candidate genes (MsFAI, MsCDPK, and MsPCO) governing alkali tolerance in alfalfa. These findings provide important candidate loci and genes for future research on alkali tolerance mechanisms and may facilitate molecular breeding of alfalfa after further functional validation.