Low-temperature stress has become an important abiotic factor affecting high and stable yield of wheat production. Therefore, it is pivotal to take appropriate measures to enhance low-temperature tolerance in wheat. In order to study the regulation of different methods of phosphorus application (MPA) on carbohydrate assimilation and grain yield formation of wheat under low-temperature stress, a pot experiment was carried out, using Yannong 19 (YN19, a cold-tolerant cultivar) and Xinmai 26 (XM26, a cold-sensitive cultivar), in the Agricultural Extraction Garden of Anhui Agricultural University (31°86′N, 117°26′E; 30 m altitude) in Hefei, Anhui Province, China. We set up optimized phosphorus application (OPA) and traditional phosphorus application (TPA) at jointing stage, then conducted chilling treatment (T1 at 4℃) and freezing treatment (T2 at -4℃) as well as at normal temperature (CK at 11℃) in an artificial climate box at the anther differentiation period, and investigated the effects of OPA and TPA on photosynthetic parameters and the accumulation and distribution of dry matter at the growth stages of booting, flowering, grain-filling and maturity. Low-temperature stress reduced plant height, tillers number and leaf areas per plant, and the above indexes of T2 treatment was significantly lower than CK treatment. The net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr) of flag leaves decreased in low-temperature treatments, whereas intercellular carbon-dioxide concentration (Ci) increased. Pn of YN19 and XM26 in T2 treatment was significantly lower than that in T1 treatment, while Pn in T1 treatment was significantly lower than that in CK at grain-filling stage. Compared with TPA, Pn of OPA treatments increased 5.2%-15.2% and 3.8%-18.7% at the same temperature level of YN19 and XM26, respectively. Moreover, low-temperature stress reduced dry matter accumulation at reproductive growth stage and dry matter transportation of vegetative organs before flowering stage. OPA increased dry matter accumulation of vegetative organs after flowering stage and promoted the transportation of assimilates to grains, so the grain number per spike (GNPS), 1000-grains weight (TGW) and yield per plant (YPP) increased. Overall, OPA can enhance low-temperature stress tolerance in wheat, effectively improve wheat architecture and photosynthesis, promote the accumulation of assimilates and transportation and distribution to grains after flowering stage, increase GNPS and TGW, and ultimately lessen yield loss. The damage of Low-temperature stress to XM26 was higher than that to YN19, and the effect of OPA was the best in T2 treatment. The purpose of this study was to improve the stress resistance of wheat and the efficient utilization and conservation of phosphorus resources.