With global warming and climate change, high-temperature stress has become a major factor affecting crop growth and yield. Cotton (Gossypium hirsutum L.) crops in the United States experience periods of extreme high temperatures during flowering and boll development, but information is lacking on the physiological response of cotton to high-temperature stress and appropriate techniques to quantify this response. Our objective was to assess the effect of high- temperature stress on physiological processes and screen a diverse set of cotton cultivars for high-temperature tolerance. Growth-room studies were conducted to investigate the effect of high temperature on photosynthesis, quantum yield of photosystem II (PSII), and membrane leakage of four obsolete and four modern cotton cultivars. The results showed that the optimum temperature for photosynthetic carbon fixation of cotton was approximately 33 °C and that photosynthesis in cotton decreased significantly at temperatures of 36 °C and above. Similarly, increasing air temperature caused a decrease in quantum yield of PSII at 36 °C and above indicating high-temperature stress. Membrane leakage was also significantly increased when temperature exceeded 33 to 35 °C. The results showed that the ‘Acala’ genotypes were more tolerant to high temperatures compared to the other genotypes. Chlorophyll fluorescence and membrane leakage were practical and reliable techniques for quantifying tolerance to high temperatures. Photosynthesis was also sensitive but was not practical for screening large numbers of genotypes for temperature tolerance.