Cotton development and yield are negatively influenced by extremes temperatures, mainly during reproductive stage. Ambient air temperature is used to evaluate temperature stress effect on yield under field conditions; however, there is evidence that actual temperatures in the canopy where bolls develop are different. In terms of cotton responses, there is limited research about the effects of elevated day and night temperatures upon boll carbohydrate content, size, and boll respiration. Field and growth chamber experiments were performed during 2014-2015 using the cotton (Gossypium hirsutum L.) cultivar DP0912B2RF. Field studies for determining temperature gradients through the canopy consisted of two planting dates with weekly measurements performed at noon, beginning one week after first flower. Temperature was recorded using thermocouple thermometers at both lower canopy (main-stem node 7+/-1) and upper canopy (main-stem node 11+/-1) positions, and included internal boll, air next to the boll, ambient air above the canopy, subtending leaf, and soil temperature. Results showed that there is a dynamic vertical temperature gradient through the canopy with the ambient air temperature being significantly cooler that the air inside the canopy. Consequently, ambient air temperature is not always an appropriate indicator of what is happening within the canopy. For analyzing the effect of high day and night temperature on boll carbohydrate and respiration, three growth chamber experiments were performed. The temperature regimes consisted of optimal temperature throughout the study (32/24°C, day/night) and high day and night temperature (38/30°C, day/night). The stress was imposed at first flower and the measurements were taken randomly two weeks later. Eight respiration measurements were recorded in a 24 hours period and boll size, boll weight, and carbohydrate content of bolls were analyzed. Results indicated that 38/30°C (day/night) temperature conditions presented a reduced boll size and a substantial decline in non-structural carbohydrate content.