Alfalfa (Medicago sativa L.) is one of the major forage legumes grown in Oman, but its productivity is significantly affected by salt stress. In this study, 20 local Omani alfalfa landraces were characterized for morphological and biochemical traits, tissue minerals, and genetic diversity. The experiment was conducted in a greenhouse with three salinity levels: control (2 dS m−1), moderate salinity stress (6 dS m−1), and severe salinity stress (10 dS m−1). Different morphological and biochemical traits and tissue minerals showed significant variation in response to varying salinity levels. With an increase in salinity stress levels from 2 to 10 dS m−1, there was a decrease in different morphological traits across the landraces, except for forage fresh weight which increased at both levels of salt stress (6 and 10 dS m−1) compared to control owing to high leaf free proline, total soluble phenolics, catalase activity, and leaf potassium concentration. However, different biochemical traits and sodium and chloride increased with the increase in salt stress levels (6 to 10 dS m−1) and the higher values were recorded at higher salt stress level (10 dS m−1). The principal component analysis (PCA) had 47.8 and 49.2% variability for morphological traits under salt stress levels of 6 and 10 dS m−1, respectively. Under both levels of salt stress, there was 81% variability by the two PCs for different biochemical traits. The total variability contributed by the two PCs for mineral nutrients was 69.2 and 66.2% for salt stress levels of 6 and 10 dS m−1, respectively. At both levels of salt stress, the landraces OMA 5, OMA 21, OMA 161, OMA 6, OMA 95, OMA 220, and OMA 285 were grouped on high morpho-biochemical traits and tissue mineral contents. There was a low to moderate level of genetic diversity (H = 0.3252) in the Omani alfalfa populations. However, the level of genetic diversity for the populations from the four regions was moderate (Dhahira) to high (Batinah, Dakiliya, and Sharqiyah). The substantial variation among the landraces on morpho-biochemical traits and tissue minerals can be used in the breeding programs to develop salt-tolerant alfalfa genotypes. The landraces OMA 6, OMA 161, and OMA 285 were excellent in performance based on morphological and biochemical traits and tissue mineral contents under both levels of salt stress.