Alfalfa (Medicago sativa L. subsp. sativa) is the most cultivated perennial forage crop worldwide. It is an autotetraploid (2n = 4x = 32), out-crossing species and it has potential to be cultivated in marginal lands affected by salinity. In this study, genetic diversity for salt tolerance was explored in 36 alfalfa genotypes from continental (15 genotypes) and coastal regions (21 genotypes), under salt stress of 150 mM NaCl. Also, characters and mechanisms that can be used to increase yield under saline conditions were investigated. All genotypes were clonally propagated into 144 seedlings (four plants/genotype) and designed in a randomized complete block under greenhouse conditions. Twelve tolerance traits were scored; fresh and dry aboveground biomasses, stem length, leaf number, [Na+], [K+], K+/Na+ ratio, catalases and superoxide dismutases, proteases, protein and reducing sugar in leaves. Stress tolerance index was calculated for each genotype. High significant variation was found among genotypes and treatments for fresh and dry matters. All genotypes had [Na+] in shoots significantly higher in stressed plants than in control plants and low K+/Na+ ratios, signifying the use of Na+ “inclusion mechanism” to prevent salt stress. The increase in catalase activity in shoots under salt condition demonstrates that this antioxidant enzyme could be playing an essential role in the tolerance mechanism. In contrast, it seems that alfalfa tolerance system to cope salt stress is not related to protease, superoxide dismutase, and proteins accumulation. Based on salt tolerance index and PCA analysis, three tolerant genotypes from coastal origin were selected that can be prominent sources for breeding program of alfalfa.