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Studies have shown that the three subunits of β-conglycinin are the main potential allergens of soybean sensitive patients. And β-conglycinin has adverse effects on nutrition and food processing. So solation and production of lines with lower β-conglycinin content has been the focus of recent soybean breeding projects. Soybean lines with deficiency in one or all subunits of β-conglycinin have been obtained. An effective and rapid system to identify such mutations will facilitate genetic manipulation of the β-conglycinin subunit composition. Here, two segregating F2 populations were developed from crosses between Cgy-1/cgy-1 (CC), an α’-lacking line (Δα’), and DongNong 47 (DN47), a wild-type (Wt) Chinese soybean cultivar with normal globulin components, and Cgy-2/cgy-2 (CB), an α-lacking line (Δα), and DN47. These populations were used to estimate linkage among the cgy-1 (conferring α’-null) and cgy-2 (α-null) loci and simple sequence repeat (SSR) markers. Seven SSR markers (Sat_038, Satt243, Sat_307, Sat_109, Sat_231, Sat_108 and Sat_190) were determined to co-segregate with cgy-1, and six SSR markers (Satt650, Satt671, Sat_418, Sat_170, Satt292 and Sat_324) co-segregated with cgy-2. Linkage maps being composed of seven SSR markers and cgy-1 locus, and six SSR markers and the cgy-2 locus were then constructed. It assigned that the cgy-1 gene to chromosome 10 at a position between Sat_307 and Sat_231, and the cgy-2 gene to chromosome 20 at a position between Satt650 and Satt671. These markers should enable map-based cloning of the cgy-1 and cgy-2 genes. For different subunit-deficiency types [α’-null, α-null and (α’+α)-null types], the two sets of SSR markers could also detect of polymorphism between three normal cultivars and seven related mutant lines. The identification of these markers is great significance to the molecular marker-assisted breeding of soybean β-conglycinin subunits.