Abstract: This research was conducted to study the effect of the north cold area on the physiological and biochemical characters of alfalfa during overwntering in order to provide theoretical basis for wide application of alfalfa. The roots of alfalfa before and after winter were used as materials. The roots were dug on 15th October, 30th October and 15th November in 2013 and 30th March, 15th April and 30th April in 2014, and then preserved in -80℃ refrigerator. Overwintering rate was surveyed in the spring of 2014. Based on analysis of main quality indexes, including soluble sugar content, soluble protein content, total nitrogen and free praline content, effects of physicochemical indexes of roots of different kinds of alfalfa were studied. The result showed that the content of soluble sugar and soluble protein, free praline of roots of‘Gongnong No.1’and‘Caoyuan No.3’were the highest among all the alfalfa varieties in the coldest month (November), followed by‘Longmu 806’and ‘Wega7F’, which had higher cold resistance ability. However, every index of roots of‘Aohan’and‘WL319 HQ’were lower than that of other varieties, their ability of cold resistance was relatively poor. Therefore, the ability of cold resistance of the six alfalfa varieties was in an order as:‘Gongnong No.1’>‘Caoyuan No.3’> ‘Longmu 806’>‘Wega7F ‘Aohan’>‘WL319 HQ’.