High temperatures (HT) over 30℃ occur frequently during the wheat grain filling season in China, and have become an important limiting factor of wheat quality. To evaluate the impact of post-anthesis HT exposure on the amino acid content and composition of wheat grains, two winter wheat cultivars of different gluten strengths, Zhengmai366 (a high-gluten-strength cultivar) and Luohan2 (a medium-gluten-strength cultivar), were investigated via a pot-culture experiment conducted at the experimental farm of Henan Agricultural University in Zhengzhou, China (113°35' E, 34°51' N). The experiment was performed using a split-plot design with two developmental stages and two durations of HT treatment. The plants were exposed to HT (38℃) in a climate-controlled greenhouse from 11:00 to 16:00 at 5 days post-anthesis (DPA), for 2 d and 4 d, and at 15 DPA for 2 d and 4 d. The control (CK) plants were treated at 28℃ in a different greenhouse. After the completion of treatment, the plants were returned to field conditions. The results were as follows:(1) post-anthesis HT significantly increased the lysine, essential amino acids (EAAs), non-essential amino acids (NAAs), and total amino acids (TAAs) content of the grains, whereas the EAA/TAA ratio and amino acid accumulation per kernel declined in both wheat cultivars. High temperatures had a stronger impact on the amino acid content of Luohan2 grains than on those of Zhengmai366. (2) The impact of HT on the amino acid content of grains was greater when HT was applied at an early stage of grain development (5 DPA) than when the treatment was applied at an intermediate stage of grain-filling (15 DPA). However, with respect to the amino acid accumulation per kernel, the effect of HT exposure at the intermediate stage (15 DPA) was greater than when the treatment was applied at the early grain-filling stage (5 DPA). (3) The amino acid content of the grains of the two wheat cultivars responded differently to different durations of HT treatment. The lysine, EAA, NAA, and TAA contents of the grains of Luohan2 significantly increased with prolonged exposure to HT. However, the lysine, EAA, NAA, and TAA contents of Zhengmai366 grains exhibited an initial decline after 2 d of HT, and then showed a significant increase after 4 d of HT. (4) The effects of the developmental stage and duration of HT exposure on the amino acid content of wheat grains differed between the two cultivars. The EAA/TAA ratio of both cultivars was strongly influenced by HT stage. With respect to amino acid content, Zhengmai366 exhibited greater sensitivity to HT duration, whereas Luohan2 exhibited a similar sensitivity to the different stages and durations of HT treatment. (5) Developmental stage and the duration of HT exposure interacted significantly to affect amino acid accumulation. During the early grain-filling stage (5 DPA), 4 d of HT exposure resulted in the lowest amino acid accumulation per kernel, whereas 2 d of treatment at the mid-grain-filling stage (15 DPA) induced the lowest amino acid accumulation per kernel, in both cultivars. In conclusion, the influence of HT stress on the content and composition of amino acids in wheat grains during the early and mid-grain-filling stages depended not only on genotype, but also on the developmental stage at which the treatment was applied, and the duration of the exposure.