To investigate the physiological mechanism of rice chalkiness formation under high temperature conditions, a pot experiment was conducted using high chalkiness trait Xiangzaoxian 24 (X24) and its parents Xiangzaoxian 11 (X11, high chalkiness trait) and Xiangzaoxian 7 (X7, low chalkiness trait) as materials. On the 6th day after heading(DAH), the rice was moved to a greenhouse for 15 days of high temperature treatment(HT), with outdoor normal temperature treatment(NT) as a control. The changes in net photosynthetic rate of flag leaves, grain filling rate, grain amylose content, and relative expression levels of starch synthesis related genes under HT was studied during the filling period. The results showed that the chalkiness of X7, X11, and X24 under HT increased by 2.53, 4.82, and 6.66 percentage points compared to the NT, respectively. High temperature caused the net photosynthetic rate of X11 and X24 to be significantly lower than that of the control on the 8th, 12th, and 16th DAH, while the net photosynthetic rate of X7 leaves was higher than that of the control on the 16th DAH. The grain filling rate of HT increased on the 8th DAH and reached its maximum value on the 12th DAH, which was 4-8 days earlier than that of the control. The analysis of gene expression differences showed that the expression levels of genes OsSSIIIa, OsAGPSL2, and GBSSI related to starch synthesis in X11 and X24 grains were significantly down-regulated on the 12th and 16th DAH, which leading a decrease in amylose content and an increase in chalkiness in rice grain.