Abstract <p>Drought at squaring and flowering significantly limits cotton (<i>Gossypium hirsutum</i> L.) production. The indeterminate growth habit of cotton makes it more vulnerable to water deficit conditions. Screening cotton cultivars for drought tolerance is crucial for sustainable production of cotton. However, the physiological alterations and metabolome profiles underlying drought tolerance/susceptibility remain unclear. To address this, the study evaluated three high yielding TNAU cotton cultivars for drought tolerance along with the TNAU drought tolerant check variety, KC3. Drought at 45% pot capacity was imposed by dry down method during squaring and flowering stages in pot culture studies. Drought tolerance in cotton was associated with increased stomatal frequency coupled with reduced stomatal size. The ability to maintain the photosynthesis and stomatal conductance with decreased transpiration rates were traits for yield protection under stress. TVH002 was identified as drought tolerant cultivar, registering comparable yields with the check variety (KC3). Metabolites in TVH002 for drought tolerance were increased synthesis of alkanes, leading to cuticular wax deposition and reduced transpiration rate and high accumulation of fatty acids, that acts as signal for membrane stability. Upregulation of secondary metabolites were found to trigger the stress defense pathways, while osmotic protection was aided by increased synthesis of amino acids and keto acids. Integration of physiological process and metabolomics data in drought stressed cotton, helped to screen TVH002 as drought tolerant cultivar. TVH007 was identified as drought susceptible cultivar. Correlation of physiological and molecular level traits with yield under drought confirmed TVH002 for drought tolerance.</p>