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In the present study, the variation in polar metabolites in Gossypium hirsutum L. during water stress was observed by nuclear magnetic resonance spectroscopy (NMR) techniques. Discriminating metabolites were elucidated by 1H NMR measurements and 2D-NMR (COSY and HSQC) techniques. Total 46 polar metabolites were detected in control and water stressed G. hirsutum leaf and stem. Nine metabolites in the leaf while thirteen metabolites in the stem were quantified. Highly significant quantitative variations were noticed, among the metabolites, i.e., glucose, glycinebetaine (GB), alanine in leaf and glycinebetaine (GB), scyloinositol in stem. This change in metabolite profile during water stress in G. hirsutum represents the change in metabolomic flux in different pathways.