To screen cold-tolerant Medicago falcata germplasms and to clarify their physiological response mechanisms to low-temperature stress， this study systematically evaluated the physiological and chlorophyll fluorescence characteristics of 20 accessions under gradient low-temperature stresses （25℃， 10℃， 5℃， 0℃）. By measuring seven key indicators， it was found that the contents of malondialdehyde （MDA）， soluble sugar （SS）， and proline （Pro）， as well as the level of initial fluorescence （F_o）， exhibited upward trends with decreasing temperature， all significantly increasing at 0℃ （P<0.05）； while the relative chlorophyll content （Soil and plant analyzer development， SPAD）， maximum fluorescence （F_m）， and maximum photochemical efficiency of PSⅡ （Maximal photochemical efficiency of photosystem II， F_v/F_m） showed downward trends. A comprehensive evaluation using the membership function method combined with the standard deviation coefficient weighting method identified M₄， M₁， and M₁₀ as accessions with stronger cold tolerance. Stepwise regression analysis further established a cold tolerance prediction model （R²=0.932）， confirming F_v/F_m and SS content as critical evaluation indices. These results provide excellent germplasm resources and a theoretical basis for cold-tolerance breeding of alfalfa， and it is recommended that future research integrate omics technologies to further elucidate the underlying molecular regulatory networks.

Medicago falcata  /  Low-temperature stress  /  Physiological response  /  Chlorophyll fluorescence  /  Cold resistance evaluation