Cotton (Gossypium hirsutum L.), a crucial global fiber and oil seed crop, faces a diverse biotic and abiotic stresses. Among these, temperature stress strongly influence its growth, prompting adaptive physiological, biochemical and molecular changes. In this study, we explored the proteomic changes underscoring the heat stress tolerance in the leaves of two locally developed cotton genotypes, i.e., heat tolerant (GH-Hamaliya Htol) and heat susceptible (CIM-789 Hsus), guided by morpho-physiological and biochemical analysis. These genotypes were sown at two different temperatures, control (35ºC) and stress (45ºC) in glass house, in randomized complete block design (RCBD) in three replications. At the flowering stage, a label-free quantitative shotgun proteomics of cotton leaves revealed the differential expression of 701 and 1270 proteins in the tolerant and susceptible genotype compared to the control, respectively. Physiological and biochemical analysis showed that the heat-tolerant genotype responded uniquely to stress by maintaining the net photosynthetic rate (Pn)( 25.2-17.5µmolCO2m-2S-1), chlorophyll (8.5-7.8mg/g FW), and proline contents (4.9-7.4 µmole/g) compared to control, supported by the upregulation of many proteins involved in several pathways including photosynthesis, oxidoreductase activity, response to stresses, translation, transporter activities as well asprotein and carbohydrate metabolic processes. In contrast, the distinctive pattern of protein downregulation involved in stress response, oxidoreductase activity, and carbohydrate metabolism was observed in susceptible plants. Thus, in this study, the specific proteins that increased in abundance under heat stress tolerance mechanism can be used as markers in future for producing the heat tolerant cotton genotypes without compromising the yield.