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Water stress is one of the major environmental stresses that affect agricultural production worldwide, especially in arid and semi-arid regions. This research investigated the effect of water deficit, induced by PEG-6000 on wheat genotypes (GA-2002, Chakwal-97, Uqab-2000, Chakwal-50 and Wafaq-2001) grown in modified MS medium solution. Osmotic stress caused a more pronounced inhibition in leaf relative water content and leaf membrane stability more sensitive (index in Wafaq-2001 and Uqab-2000) genotypes compared with the tolerant (Chakwal-50, GA-2002 and Chakwal-97) genotypes. Upon dehydration, an incline in proline, total soluble sugar, total soluble protein, superoxide dismutase, peroxidase, catalase and malondialdehyde activity content were evident in all genotypes, especially at osmotic stress of -8 bars. The observed data showed that status of biochemical attributes and antioxidant enzymes could provide a meaningful tool for depicting drought tolerance of wheat genotypes. The present study shows that genotypic differences in drought tolerance could be likely attributed to the ability of wheat plants to induce antioxidant defense under drought conditions. In order to develop genotypes with stable, higher yields in dry farming conditions, it is necessary to characterise genetic resources based on drought adaptation, determine suitable genotypes, and then use them in breeding programmes.