Low availability of phosphorus (P) is a major constraint to production of cotton (Gossypium hirsutum L.). The extent to which genotypic variation in root traits exists or contributes to P-acquisition efficiency (PAE) in cotton is unknown. To assess genetic variation in PAE, the biomass and P-acquisition characteristics of 32 cotton genotypes were evaluated in a hydroponic experiment. Significant genotypic variation in biomass and P content was detected among the cotton genotypes in two seasons. We then conducted a 2-year pot experiment to compare P-efficiency traits between three P-efficient and two P-inefficient genotypes under P-deficient and P-sufficient conditions (0 and 75 mg P₂O₅ kg^–1 soil, respectively). We detected significant differences in biomass accumulation and allocation, P accumulation and allocation, root traits and PAE among the five cotton genotypes under P-sufficient and P-deficient conditions. Compared with P-inefficient genotypes, P-efficient genotypes had longer surface fine roots, and greater total root surface area, total root length, surface root length, and P concentration (partitioning index) in bolls. Root morphology, especially surface fine root length and middle root length, played an important role in P uptake under P-deficient conditions.