Polyploid wheats include a group of tetraploids known as Timopheevii (AAGG), which are represented by two subspecies: ssp. (cultivated) and ssp. (wild). The combined use of electrophoretic (SDS-PAGE) and chromatographic (RP-HPLC) techniques carried out on high-molecular-weight glutenin subunits (HMW-GSs) permitted the association of different x- and y-type subunits to the A and G genomes and the assessment of allelic variation present at corresponding loci. The results also revealed that in both subspecies, accessions are present that possess expressed y-type subunits at the locus. Genes corresponding to these subunits were amplified and amplicons corresponding to x- and y-type genes associated with the A genome were detected in all accessions, including those without expressed x- and y-type subunits. The comparison with genes of polyploid wheats confirmed the structural characteristics of typical y-type genes, with the presence of seven cysteine residues and with hexapeptide and nonapeptide repeat motifs. The identification of wild and cultivated with both x- and y-type glutenin subunits at the and loci represents a useful source for the modification of the allelic composition of HMW-GSs in cultivated wheats with the ultimate objective of improving technological properties.