AbstractThe high-molecular-weight glutenin subunit (HMW-GS) composition of wheat is the main factor controlling gluten strength related to bread baking quality. Reported molecular markers for HMW-GS were validated and selected for improved breeding efficiency in South Korean wheat breeding programs. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, lab-on-a-chip electrophoresis, sequence-tagged site (STS) markers, and Kompetitive Allele-Specific PCR were performed to re-evaluate the known HMW-GS of 14 wheat cultivars. Glu-A1b and Glu-A1c alleles were separated by the STS marker, UMN19, and KASP marker, namely Glu-Ax1/2*_SNP, at Glu-1 loci. At the Glu-B1 locus, Glu1-By8 and Glu1-By9 could be distinguished from Glu-B1b and Glu-B1c alleles by two STS markers, namely ZSBy8 and ZSBy9a, respectively. Glu1-Bx17 and Glu1-7^OE could respectively be separated from non-Glu-B1i and non-Glu-B1al alleles by cauBx642 and BX7^OE_866_SNP. The Glu-D1d allele, used to determine bread baking quality, could easily be distinguished from other alleles by Glu-D1d_SNP at Glu-D1 loci. Validated molecular markers in this study could therefore be used to select wheat lines for good bread baking quality in South Korean wheat breeding programs.