Under the condition of pot cultivation in the field, the intelligent ultra-low temperature light incubator was used to carry out a 4h·d−1 simulation experiment of late spring coldness in the anther differentiation period of two wheat varieties, Yannong 19 (YN19, strong resistance to late spring coldness) and Xinmai 26 (XM26, weak resistance to late spring coldness). Two temperature treatments of 2℃ and −2℃ were set, and the control treatment (CK) was 10℃, in order to clarify the effect of different degrees of late spring coldness on the number of grains and grain weight at different spike positions of wheat main stem. The results showed that: (1) the number of fertile grains and grain weight at different spikelet on the main stem in different treatments were changed as a quadratic curve of first increased and then decreased. (2)Late spring coldness significantly reduced the number of fertile grains at different spikelet and grain positions of wheat main stem, and the reduction amplitude increase with the increase of stress degree. Under 2℃ and −2℃ treatment, the total fertile grains of lower, middle and upper spikelets of YN19 decreased by 40.74%, 18.75%, 35.00% and 70.37%, 32.50% and 15.00% respectively, while XM26 significantly decreased 62.50%, 7.87%, 20.00% and 100%, 42.22%, 100%, respectively. (3)Mild late spring coldness stress (2℃) reduced the grain weight of wheat main stem, while severe late spring coldness stress (−2℃) increased the grain weight due to the sharp reduction of the number of grains per spike. Compared with CK, the grain weight of spikelets of YN19 decreased by 2.93% − 47.53% and XM26 by 19.72% − 49.96% under 2℃ low temperature stress treatment; the grain weight of spikelets of YN19 increased by 20.57% − 60.84% and XM26 by 1.20% − 42.78% under −2℃ low temperature treatment. (4)The dry weight of the total grains at different parts of wheat main stem spike in two varieties was lower than that of CK due to the late spring coldness. Under the stress of 2℃ and −2℃, the dry weight of total grains of the lower and middle spikelets of YN19 significantly decreased by 49.49%, 28.99% and 65.76%, 29.47%, respectively, compared with CK, and the dry weight of total grains of the upper spikelets under −2℃was significantly reduced by 23.09% compared with CK. While under the stress of 2℃ and −2℃, the dry weight of total grains of the lower, middle and upper spikelets of XM26 significantly decreased by 69.76%, 28.61%, 20.02% and 100%, 42.33%, 100%, respectively, compared with CK. To sum up, the late spring coldness had adverse effects on the number of fertile grains and grain weight at different spikelet and grain positions of wheat main stem. The damages to the wheat main stem spike position was shown as follows: the lower spikelet > the upper spikelet > the middle spikelet, and the order of damage to the wheat grain position was shown as follows: G3 (the third grain position) > G1 (the first grain position) > G2 (the second grain position). It was concluded that the effect of late spring coldness on the number of fertile grains was greater than on the grain weight, and the degree of influence increased with the aggravation of late spring coldness stress. Both the number of fertile grains and the weight of grains showed that the lower spikelets were most affected, that is, the inferior spikelets and inferior grains were more affected by the late spring coldness. The late spring coldness during the anther differentiation period mainly reduced the number of grains per spike of the upper spikelet and the lower spikelet of the main stem spike, reduced the weight of inferior grains, and finally leads to the reduction of wheat yield. By comprehensive comparison, the damage degree of YN19 was less than that of XM26, and YN19 had stronger resistance to late spring coldness. Therefore, selecting wheat varieties with strong resistance to late spring coldness is an important measure to reduce the damage of late spring coldness in wheat production. [ABSTRACT FROM AUTHOR]