Knowledge of genetic relationships in crop breeding programs provides valuable information that can be used by plant breeders as a parental line selection tool. In Upland cotton (Gossypium hirsutum L.), the Pee Dee germplasm program represents one of the most historically significant Upland cotton breeding programs and is known as a key source of fiber quality genes for commercial cultivars. The foundation of the Pee Dee germplasm is known to represent an array of genetic diversity involving the hybridization of G. hirsutum L., G. barbadense L., and triple hybrid strains (G. arboreum L. × G. thurberi Todaro × G. hirsutum L.). In this study, we characterized genetic relationships within the Pee Dee germplasm collection using molecular marker and field performance data. Molecular marker and field performance data showed the Pee Dee germplasm collection still maintains useful amounts of genetic diversity. The methods described provide plant breeders of cotton and other crops a strategy to develop a parental line selection tool based on genotypic and phenotypic information. Cotton breeders can directly use the information provided to select specific Pee Dee germplasm parental line combinations based on genotypic (molecular marker) and phenotypic (field performance) information rather than relying on pedigree and phenotypic information alone.