Abstract: Three alfalfa cultivars with different root types (rhizomatous root of Medicago sativa L. cv. “Qingshui”; creeping root of M. varia Martin. cv. “Gannong No. 2”; and tap root of M. sativa L. cv. “Longdong”) were used to study root adaptability to low temperatures during a natural temperature increasing-decreasing process in Tianshui, Yuzhong and Wuwei Counties of Gansu Province. Dynamic variations in proline, soluble protein, soluble sugar and MDA contents, and CAT and SOD activities in the roots were determined via trace analysis in late fall, winter and spring of the following year. The results showed that variations in root MDA content increased with decreasing air temperature in late fall and early winter. The trend in MDA content then also sharply decreased with decreasing air temperature and vice versa with increasing temperature in spring of the following year. Root contents of proline and soluble sugar, and CAT activity increased with decreasing air temperature and vice versa; the rules of soluble protein and SOD activity were difference with air temperature change in different ecotopes. Membership function analysis showed the order of cold-resistance of alfalfa cultivars was as M. sativa L. cv. “Qingshui” with rhizomatous root > M. varia Martin. cv. “Gannong No. 2” with creeping root > M. sativa L. cv. “Longdong” with tap root. The order of alfalfa cold-resistance in relation to the ecotopes was Yuzhong > Wuwei > Tianshui. Alfalfa cold-resistance and altitude were positively correlated.