<p>Heat stress during flowering is a significant contributor to yield reduction in cotton. Chitosan (CH) is an environmentally friendly compound with the potential to alleviate heat stress. The purpose of the study was to assess the physico-chemical changes in cotton grown in various heat-stress conditions, and to optimize the dose of foliar-applied chitosan to alleviate the consequences of these changes. The trial was conducted in 2022 and 2023 at the research farm of Agronomy, University of Agriculture, Faisalabad, Pakistan. Heat stress treatments were applied in two ways: one was designated as control or without heat stress (NH), and the second was named as heat stress during flowering (HSF) for 4 and 8 days, whereas different concentrations (water spray, 0.2, 0.4, 0.6, and 0.8&#xa0;g L^− 1) of chitosan were foliar sprayed. Trial results showed that heat stress negatively affected different functions of plants, such as antioxidant activities, stay green (chlorophyll <i>a</i> and <i>b</i>), and water relations (water and osmotic potential), which basically resulted in a decrease in the seed cotton yield. Exogenous CH (0.6–0.8&#xa0;g L^− 1) effectively reduced the negative effects of heat stress, supported stay green and antioxidant activities, and helped raise water relations, resulting in increased seed cotton yield (16.15–21.09%) during 2022 and 2023.</p><p><?noindent??><b>Clinical Trial Number</b> Not applicable.</p>