Background: Nitrogen (N) is important for improving various morphological and physiological processes of cotton but their contribution to fiber quality is still lacking. Aims: The current study aimed to explore the relationship between root morphology, subtending leaf physiology, and fiber quality of contrasting N-efficient cotton genotypes in response to N. Methods: We analyzed the above parameters of CCRI 69 (N-efficient) and Xinluzao-30 (XLZ-30, N-inefficient) under control (2.5 mmol·L?1) and high N (5 mmol·L?1) conditions. Results: The results showed that root morphological traits were increased in CCRI-69 under control conditions than high N. Subtending leaf morphology, chlorophyll and carotenoid contents, free amino acids, and soluble proteins were higher under high N as compared with the control. However, soluble sugars, fructose, sucrose contents, and sucrose phosphate synthase were higher under control conditions than high N across the growth stages. Irrespec-tive of the N conditions, all morphological and physiological traits of cotton subtending leaf were higher in CCRI-69 than XLZ-30. Except for fiber uniformity, fiber quality traits like fiber length, strength, micronaire, and elongation were improved under control conditions than high N. Between the genotypes, CCRI-69 had significantly higher fiber length, strength, micronaire, and elongation as compared with XLZ-30. Strong positive correlations were found between root morphology, soluble sugars, sucrose content, and sucrose phosphate synthase activity with fiber qual-ity traits, respectively. Conclusions: These findings suggest that CCRI-69 performed better in terms of growth and fiber quality under rela-tively low N condition, which will help to reduce fertilizer use, the cost of production, and environmental pollution.