Soybean (Glycine max (L.) Merrill) protein quality is limited by deficiencies in the concentrations of amino acids cysteine (Cys), methionine (Met), threonine (Thr), and lysine (Lys). This study aimed to: (1) assess the effects of genotype, environment, and genotype-by-environment interactions (GEI) on seed protein quantity and quality in two recombinant inbred line (RIL) populations derived from amino acid manipulated fast neutron mutants, in multi-environment trials (MET) conducted in four environments in southern Ontario; and (2) to identify and validate quantitative trait loci (QTL) associated with seed protein quantity and quality in the two populations. Genotype and environment effects were significant for all target traits. Significant GEI effects on protein, Thr, and Lys were observed in at least one population. A modified QTL-seq analysis discovered genomic regions associated with target traits in three of the 20 soybean chromosomes. These results may be useful for breeding efforts on improving soybean protein nutrition.