Aegilops tauschii, the donor of the D genome of hexaploid wheat, is accepted as a major contributor of disease resistance and bread-making quality attributes in cultivated wheat. High molecular weight (HMW) glutenins have a significant effect on the bread-making qualities of cultivated wheat. A large range of allelic variation in 424 Ae. tauschii accessions at the Glu-D1 t locus for both x- and y-type glutenin subunits as well as at Glu-D3t was observed with SDS-PAGE using the total endosperm protein fraction. Only 4 accessions revealed more than 2 bands on SDS-PAGE. Seventeen new allelic combinations of both x- and y-type glutenin subunits at GluD-1 t and 30 new allelic profiles at Glu-D3 were detected. These combinations comprise some lines with x- or y-type null forms. RP-HPLC analysis of accession Aus 18882, which showed 5 bands when the total endosperm protein fraction was resolved on SDS-PAGE, revealed 2 x-types and 1 y-type subunit banding pattern. RP-HPLC of the gliadin fraction exhibited an omega gliadin-like subunit. SDS-PAGE processing of the gliadin-free fraction of the same accession still exhibited the gliadin-like protein. An N-terminal protein sequence of the first 7 amino acids of the slowest moving novel x-type band of accession Aus18882 indicated its uniqueness, as no entries were found to contain this internal sequence. Demonstration of novel allelic combinations at Glu-D1 and Glu-D3 loci implies the potential for exploiting Ae. tauschii to increase the genetic variability of hexaploid wheat, particularly for bread-making qualities.