Background <p>Root spatial distribution, an important root function phenotype, is closely related to drought tolerance. Drought is known to hinder cotton development, but, the effects of drought at the square stage on the spatial distribution characteristics of roots at the flower-boll and boll opening stages remain unclear.</p> Methods <p>To address this gap, 30 different cotton cultivars were grown in the field. The control treatment received routine irrigation (1175 m³·hm⁻²), and the drought treatment received reduced irrigation (822.5 m³·hm⁻²). Multiple parameters, including root traits, above-ground traits, and yield, were assessed.</p> Results <p>At the flower-boll stage, drought conditions during the square stage significantly reduced SPAD and optimal quantum efficiency (<i>F</i>_v/<i>F</i>_m) values in cotton. Cluster analysis categorized cultivars as drought-tolerant, relatively drought-tolerant, intermediate-sensitive, relatively drought-sensitive, or drought-sensitive cultivars. Under drought conditions, drought-tolerant cultivars exhibited a greater decrease in average lateral root angles, a less pronounced increase in root-shoot ratio, and 24.25% higher yield than drought-sensitive cultivars. Additionally, in the 50–60&#xa0;cm soil layer, root length density in drought-tolerant cultivars increased by 141.78%, compared to drought-sensitive cultivars under drought conditions, and the drought-sensitive and tolerance cultivars were symmetrically distributed in the 0–30&#xa0;cm soil layer. Notably, owing to substantial rainfall during the study, there were no significant differences in root spatial distribution at the boll opening stage.</p> Conclusion <p>Drought at the square stage can improve drought tolerance and reduce yield losses by reducing lateral root angles, optimizing the root-shoot ratio, and increasing root length density in deeper soil during the flower-boll stage.</p>