Upland cotton (Gossypium hirsutum) is the world's leading fiber crop and one of the most important oilseed crops. Genetic improvement of cotton has primarily focused on fiber yield and quality. However, there is an increased interest and demand for enhanced cottonseed traits, including protein, oil, fatty acids, and amino acids for broad food, feed and biofuel applications. As a byproduct of cotton production, cottonseed is an important source of edible oil in many countries and could also be a vital source of protein for human consumption. The focus of cotton breeding on high yield and better fiber quality has substantially reduced the natural genetic variation available for effective cottonseed quality improvement within Upland cotton. However, genetic variation in cottonseed oil and protein content exists within the genus of Gossypium and cultivated cotton. A plethora of genes and quantitative trait loci (QTLs) (associated with cottonseed oil, fatty acids, protein and amino acids) have been identified, providing important information for genetic improvement of cottonseed quality. Genetic engineering in cotton through RNA interference and insertions of additional genes of other genetic sources, in addition to the more recent development of genome editing technology has achieved considerable progress in altering the relative levels of protein, oil, fatty acid profile, and amino acids composition in cottonseed for enhanced nutritional value and expanded industrial applications. The objective of this review is to summarize and discuss the cottonseed oil biosynthetic pathway and major genes involved, genetic basis of cottonseed oil and protein content, genetic engineering, genome editing through CRISPR/Cas9, and QTLs associated with quantity and quality enhancement of cottonseed oil and protein.