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Low-molecular-weight glutenin subunits (LMW-GS) are a class of seed storage proteins that play a major role in the determination of the processing quality of wheat flour. The LMW-GS are encoded by multi-gene families located on the short arms of the homoeologous group 1 chromosomes, at the <i>Glu-A3</i>, <i>Glu-B3</i> and <i>Glu-D3</i> loci. Generally, more than 15 genes are present in a single bread wheat cultivar. Because of the genic complexity of these loci and the lack of efficient methods for the LMW-GS protein allele identification, the role of each LMW-GS allele in the end-use quality has not been clearly established. In the present study a PCR-based molecular marker approach has been used to identify the LMW-GS gene alleles present in different hexaploid wheat cultivars. Analysis of a set of standard wheat cultivars with known LMW-GS alleles revealed the potential of this marker system for the unambiguous identification of the LMW-GS protein alleles. Moreover, the application of this system to three different populations segregating for the LMW-GS gene alleles showed the presence of intra-locus recombination suggesting a possible revision of the concept of a “single locus” for all the three <i>Glu-3</i> loci. The results of this study will help to define the LMW-GS profile of different hexaploid wheat cultivars and to clarify the contribution of each LMW-GS gene alleles in the control of the end-use quality.