Cottonseeds, rich in high-quality protein and fatty acids, represent a vital plant-derived feedstuff and edible oil resource. To systematically investigate genetic variation patterns in nutritional quality and screen superior germplasm, this study analyzed 26 nutritional quality traits and 8 fiber traits across 259 upland cotton (Gossypium hirsutum L.) accessions using multivariate statistical approaches. Results revealed significant genetic diversity in cottonseed nutritional profiles, with coefficients of variation ranging from 3.42% to 26.37%. Moreover, with advancements in breeding periods, the contents of protein, amino acids, and the proportion of unsaturated fatty acids (UFAs) increased, while oil content and C16:0 levels decreased. Correlation analyses identified significant positive associations (p < 0.05) between proteins, amino acids, UFAs, and most fiber traits, except for seed index (SI), fiber micronaire (FM), and fiber elongation (FE). Through a principal component analysis–fuzzy membership function (PCA-FMF) model, 13 elite accessions (F > 0.75) with high protein content, high UFA proportion, and excellent fiber quality were identified. These findings provide both data-driven foundations and practical germplasm resources for value-added utilization of cottonseed and coordinated breeding for dual-quality traits of nutrition and fiber.