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Freezing injury is one of the major disasters for the production of winter wheat in the North China Plain, which leads to a significant decrease of wheat yield. This study was conducted to investigate the impact of subfreezing temperature on freezing injury of various winter wheat cultivars at different sowing time. Three wheat cultivars, including Zhengmai 9023, Wanmai 48 and Wanmai 50, were sowed on 25 September and 5 October, respectively. Plant anatomy was applied to investi- gate the impact of subfreezing temperature on cells of three wheat cultivars, results showed that severe plasmolysis occurred in wheat sowed earlier suddenly encoun- tered subfreezing temperature without cold acclimation, compared with wheat sowed at proper sowing time. The degree of plasmolysis of different cultivars and tissues of wheat had significant differences and showed positive correlation with subfreezing temperature. Wanmai 50 had the highest cold resistance compared with Zhengmai 9023 and Wanmai 48, and there was no significant difference between Zhengmai 9023 and Wanmai 48. This study concluded that wheat cold resistance may be im- proved by adopting proper cultivars and sowing dates.