Protein and sugar content are important seed quality traits in soybean because they improve the value and sustainability of soy food and feed products. Thus, identifying Quantitative Trait Loci (QTL) for soybean seed protein and sugar content can benefit plant breeders and the soybean market by accelerating the breeding process via marker-assisted selection. For this study, a population of recombinant inbred lines (RILs) was developed from a cross between R08-3221 (high protein and low sucrose) and R07-2000 (high sucrose and low protein). Phenotypic data for protein content were taken from the F2:4 and F2:5 generations. The DA7250 NIR analyzer and HPLC instruments were used to analyze total seed protein and sucrose content. Genotypic data were generated using analysis via the SoySNP6k chip. A total of four QTLs were identified in this study. Two QTLs for protein content were located on chromosomes 11 and 20, and two QTLs associated with sucrose content were located on chromosomes 14 and. 11, the latter of which co-localized with detected QTLs for protein, explaining 10% of the phenotypic variation for protein and sucrose content in soybean seed within the study population. Soybean breeding programs can use the results to improve soybean seed quality.