BackgroundAlfalfa (Medicago sativa) is the worldwide major feed crop for livestock. However, forage quality and productivity are reduced by salt stress, which is a common issue in alfalfa-growing regions. The relative salt tolerance is changed during plant life cycle. This research aimed to investigate the relative salt tolerance and the underlying mechanisms of two alfalfa varieties at different developmental stages.ResultsTwo alfalfa varieties, "Zhongmu No.1 (ZM1)" and "D4V", with varying salt tolerance, were subjected to salt stress (0, 100, 150 mM NaCl). When the germinated seeds were exposed to salt stress, D4V exhibited enhanced primary root growth compared to ZM1 due to the maintenance of meristem size, sustained or increased expression of cell cycle-related genes, greater activity of antioxidant enzymes and higher level of IAA. These findings indicated that D4V was more tolerant than ZM1 at early developmental stage. However, when young seedlings were exposed to salt stress, ZM1 displayed a lighter wilted phenotype and leaf cell death, higher biomass and nutritional quality, lower relative electrolytic leakage (EL) and malondialdehyde (MDA) concentration. In addition, ZM1 obtained a greater antioxidant capacity in leaves, indicated by less accumulation of hydrogen peroxide (H2O2) and higher activity of antioxidant enzymes. Further ionic tissue-distribution analysis identified that ZM1 accumulated less Na+and more K+in leaves and stems, resulting in lower Na+/K+ratio, because of possessing higher expression of ion transporters and sensitivity of stomata closure. Therefore, the relative salt tolerance of ZM1 and D4V was reversed at young seedling stages, with the young seedlings of the former being more salt-tolerant.ConclusionOur data revealed the changes of relative order of salt tolerance between alfalfa varieties as they develop. Meristem activity in primary root tips and ion transferring at young seedling stages were underlying mechanisms that resulted in differences in salt tolerance at different developmental stages.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04335-3.