Soybean [Glycine max (L.) Merr.] cultivars with elevated concentrations of the α′ subunit of β-conglycinin (BC) may provide health benefits to soy protein consumers. Two Monsanto single nucleotide polymorphism markers were used to classify F2 plants in four segregating populations as having elevated α′ concentration (EAP) or normal α′ concentration (NAP). Seeds from one F3 progeny of each F2 plant were analyzed for protein composition by sodium dodecyl-sulfate polyacrylamide gel electrophoresis. Of the 800 plants in the four populations evaluated with the two markers, 82% were correctly classified for the EAP trait and 80% for the NAP trait. The EAP and NAP traits were evaluated in combination with the four glycinin genotypes gy1,2, Gy3, gy4, Gy5; gy1,2, Gy3, gy4, gy5; gy1,2, gy3, gy4, Gy5; and gy1,2, gy3, gy4, gy5. The seed yield, time of maturity, and protein and oil concentration were evaluated for 18 F3:5 lines in each of the eight genotypic classes of the four populations at three environments in 2011. The EAP trait resulted in a significant increase in the α′ and β subunits of BC and a decrease in the α subunit, with no consistent impact on total BC, total glycinin, seed yield, maturity, and protein and oil concentrations. It should be possible to develop acceptable soybean cultivars that have the EAP trait in combination with the glycinin genotype gy1,2, gy3, gy4, gy5 to obtain a high concentration of the α′ subunit and total BC with no glycinin for soy-based food products.