Abstract: The dynamic change of soluble sugar content, total nitrogen, malondialdehyde, free proline and free amino acid in root of three alfalfa (Medicago sativa) varieties were analyzed to find out their relationships with cold resistance of alfalfa. The results indicated that the content of soluble sugar, total nitrogen, free proline and free amino acid increased from late autumn and early winter, and kept higher level during the entire winter, then decreased in regreen time in the next spring, the content of malondialdehyde increased in autumn and decreased with the falling of temperature in late autumn. Moreover, the integrated determination parameters of all alfalfa varieties suggested that the coldresistance of Algonquin was the best, Gold Empress took the second place, and Gannong No.3 was the worst.