PCR-based assays with co-dominant or dominant allele-specific STS (sequence tagged sites) markers and sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) have been used in this study to discriminate Glu-1 and Glu-3 alleles in Korean wheat cultivars. Three alleles were identified at each high molecular weight glutenin (Glu-1) homoeologous locus and 10 alleles were identified at low molecular weight glutenin (Glu-3) homoeologous locus in Korean wheat cultivars. At Glu-1 loci, wheat cultivars contained Glu-A1a, b and c alleles amplified a 1093, 1063 and 920bp fragments, respectively, Glu-B1b, c and f alleles discriminated with 707(Glu-B1b and f) and 662bp(Glu-B1c) fragments and Glu-D1a, d and f alleles amplified a 576(Glu-D1d) and 612bp(Glu-D1a and f) fragments, respectively. At Glu-3 loci, wheat cultivars contained Glu-A3c, d and e alleles amplified a 573, 967 and 158bp fragments, respectively. Glu-B3d, g, h and i alleles gave a 662, 853, 1022 and 621bp, respectively and Glu-D3a, b and c alleles amplified a 884(Glu-D3a and b) and 338bp(Glu-D3c) fragments. A multiplex-PCR assay was also established which permitted the discrimination of the major Glu-1 and Glu-3 to determine glutenin compositions in a single PCR reaction and agarose gel assay because these systems could be useful to select DNA-based identification of wheat lines carrying good breadmaking performance in Korean wheat breeding programs. Three multiplex-PCR for Glu-1 and Glu-3, Glu-A1c + Glu-B1acf + Glu-D1d, Glu-A3ac + Glu-A3d + Glu-A3e and Glu-B3d + Glu-B3fg + Glu-B3h were established in this study.