Abstract: Providing a theoretical basis for drought-resistant breeding and stress-resilient cultivation techniques. This study employed drought-insensitive (YZ 05-51) and drought-sensitive (YT 93-159) sugarcane varieties as materials. During the elongation stage, we measured various agronomic traits (fresh and dry weights of shoots and roots, leaf area, net photosynthetic rate, intercellular CO₂ concentration, stomatal conductance, transpiration rate, chlorophyll content, leaf relative water content), along with antioxidant enzyme activities and osmotic adjustment substances in leaves and roots, and leaf/root tip ultrastructure under drought stress (30%±5% soil water content) compared to control (70%±5% soil water content) conditions. Correlation and grey relational analyses were utilized to examine the relationships between agronomic traits, physiological and biochemical traits, and the drought resistance index. Under drought stress, the reductions in shoot dry weight, root dry weight, net photosynthetic rate, and transpiration rate in YT 93-159 (50.09%, 29.93%, 50.82%, 47.14%, respectively) were significantly greater than those in YZ 05-51 (30.86%, 11.05%, 38.82%, 36.12%, respectively) (P<0.05). Grey relational analysis indicated that root ascorbate peroxidase (APX) activity, soluble sugar content, and malondialdehyde (MDA) content exhibited the strongest association with drought resistance. Ultrastructural observations revealed vacuole marginalization in the leaves of both varieties under water stress. YT 93-159 exhibited disintegration of grana lamellae in chloroplasts and pronounced plasmolysis in root tip cells. In contrast, YZ 05-51 maintained chloroplast grana integrity and greater plasma membrane stability in root tip cells. The drought-sensitive variety YT 93-159 displayed more significant stress-induced damage characteristics concerning photosynthetic system stability, cell membrane integrity, and osmotic adjustment capacity. Drought stress induced the accumulation of peroxides and osmotic adjustment substances in sugarcane leaves and roots. It regulated cellular ultrastructure through effects on membrane stability and intracellular osmotic pressure. Differences in the accumulation of peroxides and osmolytes between varieties with contrasting drought sensitivity contribute to their differential cellular homeostasis, manifesting as differences in drought tolerance. Root osmotic adjustment substances, particularly soluble protein content, ascorbate peroxidase (APX) activity, and malondialdehyde (MDA) content, showed high correlation coefficients with drought tolerance and may serve as key physiological indicators for screening drought-tolerant sugarcane germplasm.