Alfalfa (Medicago sativa L.), when exposed to abiotic stress such as salinity, suffers significant losses in yield and productivity. The present study evaluated the salinity tolerance of 12 alfalfa cultivars in vitro using five concentrations of sodium chloride (NaCl), ranging from 0 to 250 mmol L-1. The results obtained in the current study revealed that the Saudi cultivars, Kasimi and Hassawi, and the German cultivar (Berlin) had the highest salinity tolerance in terms of germination percentage (GP), corrected germination rate index (CGRI), days to reach 50% germination (GT(50)), and ability to form cotyledonary and true leaves. Under mmol L-1 NaCl, the Saudi cultivar Kasimi cultivar showed GP, CGRI, and GT(50) of 55.20%, 123.15, and 3.77 days, respectively. Similarly, the German cultivar (Berlin) showed GP, CGRI, and GT(50) of 50.06%, 86.61, and 5.17 days, respectively. These findings might reveal a pivotal aspect in salt tolerance in alfalfa. Our results will help to select salt-tolerant alfalfa cultivars that could thrive in arid and semi-arid areas with salinity problems.