Cold stress is one of the main abiotic stresses faced by winter wheat, which results in significant yield loss, especially in the harsh winter of the Heilongjiang province. Glycine betaine (GB), an important osmolyte in higher plants, helps in the stabilization of the plasma membrane and its protection from cold stress. In the present study, two winter wheat varieties that differ in cold resistance, Dongnongdongmai1 (DM1) and Jimai22 (J22), were planted under natural conditions and used for analyzing relative electrical conductivity, malondialdehyde (MDA) content, betaine aldehyde dehydrogenase (BADH) activity, expression of BADH, and the content of GB. The cold-resistant variety, DM1, showed a greater increase in BADH activity and GB content and decrease in MDA content than J22 under freezing conditions. GB was observed to have an obvious role in inhibiting the MDA content. This was reflected by the expression of BADH and enhanced tolerance to cold stress upon GB accumulation, which helped membrane stabilization. The results of the present study confirmed the role of GB in conferring cold resistance in the DM1 winter cultivar and could benefit other studies aimed at improving the tolerance of other wheat cultivars and other crops.