Cotton (Gossypium spp.), as an important dual-use crop, provides natural fiber to the textile industry and nutrition components for both humans and livestock. Genetic studies with cottonseed are often limited although it should be possible to improve oil and protein content while maintaining planting seed quality without sacrificing lint production. Therefore, a better understanding of the genetics and breeding of cottonseed traits and their relationship to lint production should be worthy of investigation. In this study, protein and oil contents were investigated for 13 chromosome substitution lines, their recurrent parent TM-1 and donor parent 3-79, and five commercial cultivars in four environments. We determined the genetic variations of cottonseed oil and protein levels among these lines. In addition, we determined chromosomal association with seed nutritional and quality components with the use of chromosome substitution lines. This study will provide information to guide breeders' decisions as to the feasibility of improving seed oil and protein, and other seed traits while also improving lint yield and fiber quality.