To screen Medicago varia breeding materials with strong cold resistance and explore the physiological response mechanisms of their root systems under low-temperature stress, 21 Medicago varia germplasm accessions were selected in this study, with two cold-resistant Medicago sativa cultivars(Gongnong No.1 and Gongnong No. 5) as controls. Under low-temperature stress conditions, the contents of catalase(CAT), peroxidase (POD), superoxide dismutase(SOD), malondialdehyde(MDA), free proline(Pro), and soluble sugar(SS) in the root crown of Medicago varia were measured. A comprehensive evaluation of the low-temperature tolerance of Medicago varia roots was conducted using principal component analysis and membership function analysis. The results showed that under low-temperature stress during the overwintering period, the variation coefficients of the six physiological indicators in Medicago varia roots ranged from 16.71% to 39.37%. Through principal component analysis and membership function evaluation, nine Medicago varia accessions with good cold resistance were identified. Furthermore, a regression model was constructed using different physiological indicators as dependent variables and the comprehensive evaluation value D as the independent variable. Cluster analysis of the D values divided the materials into three major groups. This study provides theoretical basis and reference for the breeding of cold-resistant alfalfa varieties.