Purpose <p>Drought stress, as one of the major abiotic stresses, has been expanded by global warming, with negative effects on cotton seed potential, cotton fiber, plant growth and yield. Therefore, many plant species have activated a drought stress memory on the physiological and biochemical level to increase drought stress threshold by balancing carbohydrate and starch and reducing water loss.</p> Methods <p>The experiment in this study was carried out to improve our understanding of water-shortage memory in cotton. Golestan cotton genotype was planted in the double and triple water-stress exposures using split-plot factorial design. There were four levels of water requirements (rain-fed, 33%, 66%, and 100% Field capacity (FC).</p> Results <p>Bioinformatics results showed that catalase enzyme (CAT) with regulatory elements and Abscisic acid regulatory element (ABRE) play a role in abiotic stresses. On the other hand, Stress Tolerance Indices (STI), Yield Stability Indices (YSI), glucose content of the leaves and early-maturity of the seeds in S₃₃ and starch content of S₂₃ were the highest among the corresponding indices resulted from other seed treatments. Among fiber quality traits, fiber length and fiber strength of seeds planted in the triple water-stress exposure were greater than those in the double water-stress exposure. In addition, seed reproduction under triple water-stress exposure at 33% field capacity (FC) demonstrated higher STI and YSI than under double water-stress exposure.</p> Conclusions <p>This suggests that plants can develop stress memory to enhance drought tolerance by activating catalase and balancing starch and glucose concentrations. Therefore, advanced breeding techniques that focus on memory responses to water scarcity can help address the challenges associated with drought.</p>