Technical Abstract: High molecular weight (HMW) glutenin subunits (GSs) play an important role in determining dough viscoelastic properties and end-use quality in cultivated wheat, and they are also excellent protein markers for genotype identification. The HMW-GSs in wheat species (Triticum ssp.) and Aegilops tauschii have been extensively characterized, but they have not been investigated in most other wild relative species. In this study, we analyzed the HMW-GSs of Thinopyrum bessarabicum, Th. intermedium, Lophopyrum elongatum, Ae. markgrafii and 43 disomic addition lines derived from crosses of these wild species with common or durum wheat using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and urea/SDS-PAGE. A total of 20 novel alleles for HMW-GSs were identified from the alien species. Among the eight sets of addition lines, only four addition lines carried the HMW-GSs derived from alien species, indicating that addition lines for group 1 alien chromosomes are absent in four sets. In the wheat-Th. intermedium chromosome 1Ai addition line, TA3649, the HMW glutenin subunits encoded by the Glu-D1 locus were missing while the alien HMW-GSs were present, indicating that the Glu-D1 locus might be lost or the presence of alien chromosomes might silence the expression of the Glu-D1 alleles. The wheat-Th. intermedium chromosome 7Ai long arm ditelosomic addition line, TA3659, carried two sets of HMW GSs (4+12 and 5+10) encoded by Glu-D1 locus. The results from this study also provided information on the alien chromosome donor accessions and wheat parents for some disomic addition lines. Such information is necessary for the development of molecular markers that will facilitate the introgression of desirable genes from the alien chromosomes into wheat genomes. The novel HMW-GSs alleles identified in this study may serve as new genetic resources for wheat quality improvement.