The increased production of reactive oxygen species (ROS), possibly leading to damage to intracellular homeostasis, is a response to temperature stresses. Hydrogen peroxide in high concentrations results in oxidative stress and even causes cell death; it acts as a signalling molecule and increases the activities of antioxidant enzymes due to gene expression in low concentrations. At the same time, H2O2 is the most stable ROS and acts as a key regulator of some physiological and biochemical processes. Consequently, the work was performed to study the stimulating effect of pretreatment with H2O2 (10mM) in 6 cotton cultivars exposed to high temperatures (45°C). The findings from the study clearly showed an increase in the activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), as well as of proline concentrations in the cotton sprouts exposed to heat stress, as compared to not pretreated ones. Pretreatment with 10mM H2O2 improved tolerance to oxidative stress arising under heat stress. The extent of lipid membrane damage was low in the heat-resistant and heat-tolerant cotton cultivars. Spraying with 10mM H2O2 is thought to cause accumulation of intracellular hydrogen peroxide before exposure to stress; it's further increased quantity as a signalling molecule led to the expression of genes of protective proteins of the antioxidant system and osmolytes (proline), providing the balance between the accumulation and removal of free oxygen radicals.