Low-molecular-weight glutenin subunit (LMW-GS) Glu-B3 has a significant influence on the processing quality of the end-use products of common wheat. To characterize the LMW-GS genes at the Glu-B3 locus, gene-specific PCR primers were designed to amplify eight near-isogenic lines and Cheyenne with different Glu-B3 alleles (a, b, c, d, e, f, g, h and i) defined by protein electrophoretic mobility. The complete coding regions of four Glu-B3 genes with complete coding sequence were obtained and designated as GluB3-1, GluB3-2, GluB3-3 and GluB3-4. Ten allele-specific PCR markers designed from the SNPs present in the sequenced variants discriminated the Glu-B3 proteins of electrophoretic mobility alleles a, b, c, d, e, f, g, h and i. These markers were validated on 161 wheat varieties and advanced lines with different Glu-B3 alleles, thus confirming that the markers can be used in marker-assisted breeding for wheat grain processing quality.