Technical Abstract: End-use quality is one of the priorities of modern wheat breeding. Even though quality is a complex trait, high molecular weight (HMW) glutenins play a major role in determining the bread making quality of wheat. DNA markers developed from the sequences of HMW glutenin genes were reported in several previous studies to facilitate marker-assisted selection (MAS). However, most of the previously available markers are dominant and amplify large DNA fragments, and thus are not ideal for genotyping using modern equipment. The objective of this study was to develop and validate co-dominant markers suitable for high throughput MAS for HMW glutenin subunits encoded at the Glu-A1 and Glu-D1 loci. Indels were identified by sequence alignment of allelic HMW glutenin genes, and were targeted to develop locus-specific co-dominant markers. Marker UMN19 was developed by targeting an 18-bp deletion in the coding sequence of subunit Ax2*. A single DNA fragment was amplified by marker UMN19, and was placed onto chromosome 1AL. Sixteen wheat cultivars of known HMW glutenin subunits were used to validate marker UMN19. The cultivars with subunit Ax2* amplified the 362-bp fragment as expected, and the 344-bp fragment was observed for cultivars with subunit Ax1 or the Ax-null allele. Two co-dominant markers UMN25 and UMN26 were developed by targeting the fragment size polymorphic sites between subunits Dx2 and Dx5, and between Dy10 and Dy12, respectively. The 16 wheat cultivars with known HMW glutenin subunit composition were genotyped with markers UMN25 and UMN26, and the genotypes perfectly matched their subunit types. Four F2 populations segregating for the Glu-A1 or Glu-D1 loci were successfully genotyped with UMN19, UMN25, and UMN26 using fluorescent labeling of PCR products separated and analyzed with the Applied Biosystems 3130xl Genetic Analyzer.