Current conditions of 2–11 kJ m−2 day−1 of UV-B radiation and temperatures of >30°C during flowering in cotton cultivated regions are projected to increase in the future. A controlled environment study was conducted in sunlit growth chambers to determent the effects of UV-B radiation and temperature on physiology, growth, development and leaf hyperspectral reflectance of cotton. Plants were grown in the growth chambers at three day/night temperatures (24/16° C, 30/22° C and 36/28° C) and three levels of UV-B radiation (0, 7 and 14 kJ m−2 day−1) at each temperature from emergence to 79 days under optimum nutrient and water conditions. Increases in main stem node number and the node of first fruiting branch and decrease in duration to first flower bud (square) and flower were recorded with increase in temperature. Main effects of temperature and UV-B radiation were significant for net photosynthetic rates, stomatal conductance, total chlorophyll and carotenoid concentrations of uppermost, fully expanded leaves during squaring and flowering. A significant interaction between temperature and UV-B radiation was detected for total biomass and its components. The UV-B radiation of 7 kJ m−2 day−1 reduced boll yield by 68% and 97% at 30/22°C and 36/28° C, respectively, compared with yield at 0 kJ m−2 day−1 and 30/22° C. No bolls were produced in the three temperature treatments under 14 kJ m−2 day−1 UV-B radiation. The first-order interactions between temperature, UV-B radiation and leaf age were significant for leaf reflectance. This study suggests a growth- and process-related temperature dependence of sensitivity to UV-B radiation.