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In the context of global warming, low temperature (LT) disasters in major crops are also becoming more common. LT stress in the Huang-Huai-Hai Plain, the central wheat region in China, caused a massive reduction in wheat yields. A step towards ensuring wheat yield stability and food security, this study investigated the effects of optimizing phosphorus application on the root physiology, dry matter phosphorus accumulation, translocation, and partitioning of wheat under LT stress, using the representative cultivar Yannong 19 as the test material. The treatments included conventional phosphorus application (R1) and twice-split phosphorus application (R2), followed by −4 °C LT treatment and normal temperature (NT) treatment during the anther interval stage. Analysis of the root physiology (enzymatic activities and acid phosphatase, contents of malondialdehyde, soluble sugar, and soluble protein), phosphorus and dry matter accumulation, translocation, partitioning, and agronomic and yield-related components was carried during this research study. The results showed that the wheat root activity was significantly reduced and the antioxidant enzyme activities were increased to mitigate the damage of LT stress. Moreover, LT treatments damaged root function. The root activity and antioxidant properties were significantly lower than those of the NT treatment at the flowering stage. The dry matter and phosphorus accumulations were reduced by 30.6~33.6% and 15.1~21.3% at the flowering and maturity stages, resulting in final yield losses of 10.3~13.0%. In contrast, root activity increased by 16.1~27.2% in the twice-split phosphorus application treatments, and the root antioxidant characteristics were higher. As a result, dry matter and phosphorus accumulation increased after twice-split phosphorus application and their translocation to the grains was more; the final yield increased by 5.5~7.3%. Overall, the twice-split phosphorus application enhanced the physiological function of the root system and promoted the accumulation of nutrients and their transport to the grain, and alleviated the yield loss of wheat caused by LT stress. [ABSTRACT FROM AUTHOR]