AbstractCotton (Gossypium hirsutum L.) cultivars exhibit varying responses to heat stress. To investigate the heat resistance of various cotton and establish an index system for evaluating their heat resistance, 21 cotton cultivars were selected and subjected to two temperature regimes (CK, average temperature 28°C, 32/24°C; HT, average temperature 38°C, 42/34°C). The results showed that under high temperatures, different changes occurred in individual indexes of cotton, reflecting the differences in heat resistance in cotton cultivars. A total of 21 cotton cultivars could be classified into four types: heat‐tolerant, moderate heat‐tolerant, moderate heat‐sensitive and heat‐sensitive cultivars by multivariate statistical analysis. Moreover, the indexes of net photosynthetic rate (Pn), leaf superoxide dismutase activity (LSOD), the maximum photochemical quantum yield (Fv/Fm), the actual photochemical quantum yield (ΦPSII), leaf malondialdehyde content (LMDA), leaf catalase activity (LCAT), dry matter weight of shoot (SDW) and root malondialdehyde content (RMDA) were determined to be useful for evaluating the cotton heat tolerance by stepwise regression analysis. The pot experiment showed that the reduction of boll number, boll weight and seed cotton yield was more remarkable under HT in the heat‐sensitive cultivar CCRI‐92 than in the heat‐resistant cultivar CCRI‐69, which further verified the screening results. In conclusion, the heat‐sensitive cultivars CCRI‐92 and heat‐resistant cultivar CCRI‐69 which are identified by seedling experiment could serve as ideal experimental materials for studying heat resistance in cotton. The physiological indices such as Pn, LSOD, Fv/Fm, ΦPSII, LMDA, LCAT, SDW and RMDA be employed for assessing the heat tolerance in cotton.