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Soybean (Glycine max [L.] Merr.) seed is about 40% protein, most of which is accounted for by the major storage proteins β-conglycinin (7S globulin) and glycinin (11S globulin). β-conglycinin, which consists of three subunits, α, α', and β, is the main allergen in soybean. Accordingly, elimination of β-conglycinin from seed is one of the goals of soybean breeding programs. Soybean accessions PI 200485 and CS 1150 lack the α'- and α-subunits of β-conglycinin, respectively. In this study, we developed a 3-betacon marker that could simultaneously identify genotypes for the three subunit genes of β-conglycinin. Interestingly, the three subunit genes were amplified with three distinct bands representing varied amplicon sizes. Mutant alleles could be identified in progeny of a three-way cross, and the corresponding α- and α'-subunit proteins were clearly absent in each mutant accession. 3-betacon marker will be highly useful for soybean breeding programs to develop soybean cultivars with decreased β-conglycinin contents. The online version of this article (doi: 10.5073/JABFQ.2015.088.041) contains supplementary files.