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Present work was aimed at studying the variation of non polar metabolites content in Gossypium hirsutum L. under water stress condition using a gas chromatography-mass spectrometry (GC-MS) technique. A total of 17 non-polar metabolites were detected in control and water stressed G. hirsutum leaf. The major metabolites were quinoline derivative (26.37±0.29%), 2- methylhexadecan-1-ol (7.47±0.07%), phytol (7.71±0.02%), myristic acid (5.94±0.04%), hexadecanol (14.30±0.94%), nonadecane (1.67±0.05%) and palmitic acid (3.20±1.39%). Fourteen metabolites were detected in control and water stressed G. hirsutum stem. The major metabolites were dodecene (1.67±0.11%), L-lysine (0.65±0.06%), dibutylphthalate (5.06±1.88%), linoleic acid (10.26±0.07%), campesterol (0.87±0.04%) and stigmasterol (1.13±0.55%). Statistical analysis of GC-MS data was carried out by Mann-Whitney U test without normal distribution using statistical software SYSTAT version 12.0. Significant variation in the content of the most of the metabolites were observed between control and water stressed leaf or stem (Mann-Whitney U test, P =0.05). It concludes that the major metabolites played an important role during water stress and can be consider as metabolites responsible for water stress tolerance in G. hirsutum.