Abstract: Eleven alfalfa(Medicago sativa L.)cultivars , which level of fall dormancy is from 2 to 9 were selected and planted in pots in this experiment. Every alfalfa cultivars was divided into six groups(a control and five treatments) randomly. The effect of low temperature stress and alfalfa cultivars on the survival rate, physiological and biochemical indexes of alfalfa seedlings was studied. The results showed that the effect of low temperature stress, alfalfa cultivars and interaction between them on the survival rate and physiological and biochemical indexes of alfalfa seedlings was great significant(P<0.01). For the seedlings having two cotyledons ,when they were exposed to the temperature of -7.5℃ about 12h，the survival rate was only 3.26%. when they were exposed to the temperature of -5℃ about 12h，the survival rate was 54.73% and free proline content, POD activity, membrane relative penetration and root vitality in the leaves great significant(P<0.01) increased than the control. If the seedlings were exposed to the temperature of -5℃ about 2-3h per day for 7 days, survival rate would great significant(P<0.01)increased (> 95%). Free proline content, soluble sugar, soluble protein, SOD activity, root vitality and membrane relative penetration in the leaves also showed great significant(P<0.01) increased. Result also showed that for the survival rate, the cultivars which level of fall dormancy is from 4 to 9 appeared great significant(P<0.01)lower than those which level of fall dormancy is from 2 to 3. And the similar results occurred in the free proline content, POD activity and membrane relative penetration.