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Root system architecture (RSA) plays an important role in soil water uptake and plant growth. However, there is a lack of understanding regarding the connection between the changes in RSA characteristics under drought stress in cotton plants and their drought tolerance and yield. This study aims to evaluate the RSA variations in different drought-tolerant cotton cultivars under drought stress, and to assess the correlation between RSA, drought tolerance, and yield. Three drought-tolerant cotton cultivars and three drought-sensitive cotton cultivars were grown in a field equipped with rain shelters to prevent interference from rainfall. Drought stress was initiated at the three-leaf stage, maintaining the soil relative water content at 50 ± 5%, while control plants were irrigated normally at 75 ± 5% soil water content. Multiple parameters, including leaf water potential, relative water content, seed cotton yield, and RSA traits, were assessed. Notable variations in drought tolerance were observed among different cultivars when exposed to drought stress. Specifically, drought-tolerant cultivars exhibited a 34% increase in the average length of all lateral roots and a 15% increase in maximum root depth under drought stress. In contrast, seed cotton yield experienced a reduction of 22.34% in such conditions. Interestingly, there were significant differences in several RSA traits under drought stress, which were not evident under well-watered. Leaf water potential and relative water content were positively correlated with specific root length, dry root weight, average length of all lateral roots and maximum depth, and negatively correlated with average lateral root emergence angle and width/depth ratio. These results underscore the close association between RSA traits and plant drought tolerance. There was also a strong correlation between the seed cotton yield and RSA traits. Specifically, seed cotton yield increased linearly with specific root length, average lengths of lateral roots, and maximum depth, but decreased linearly with root tissue density, and average lateral root emergence angle and width/depth ratio. Optimizing RSA improves drought tolerance and reduces yield loss in drought-tolerant cotton cultivars. Understanding of the role of RSA in plant adaptation to drought stress is important for selecting and developing high-yielding cultivars with superior drought tolerance. This knowledge holds great significance in improving cotton resilience and facilitating adapting to abiotic stress through genetic improvement or agronomic measures. • Genotypic variation in root system architecture (RSA) was revealed under drought. • Root system architecture plays important roles in cotton drought tolerance. • There is a significant coefficient of variation in RSA under drought. • Optimizing RSA can improve cotton drought tolerance and minimize yield loss under drought.. [ABSTRACT FROM AUTHOR]