The wheat seed storage proteins gliadin and glutenin are encoded by multigenes. Gliadins are further classified into α-, γ-, δ- and ω-gliadins. Genes encoding α-gliadins belong to a large multigene family, whose members are located on the homoeologous group 6 chromosomes at the Gli-2 loci. Genes encoding other gliadins are located on the homoeologous group 1 chromosomes at the Gli-1 loci. Two-dimensional polyacrylamide gel electrophoresis (2-DE) was used to characterize and profile the gliadins. The gliadins in aneuploid Chinese Spring wheat lines were then compared in this study. Gliadin proteins separated into 70 spots after 2-DE and a total of 10, 10 and 16 spots were encoded on chromosomes 6A, 6B and 6D, respectively, which suggested that they were α-gliadins. Similarly, six, three and seven spots were encoded on chromosomes 1A, 1B and 1D, respectively, which indicated that they were γ-gliadins. Spots that could not be assigned to chromosomes were N-terminally sequenced and were all determined to be α-gliadins or γ-gliadins. The 2-DE profiles showed that specific α-gliadin spots assigned to chromosome 6D were lost in tetrasomic chromosome 2A lines. Furthermore, western blotting against the Glia-α9 peptide, an epitope for celiac disease (CD), suggested that α-gliadins harboring the CD epitope on chromosome 6D were absent in the tetrasomic chromosome 2A lines. Systematic analysis of α-gliadins using 2-DE, quantitative RT-PCR and genomic PCR revealed that tetrasomic 2A lines carry deletion of a chromosome segment at the Gli-D2 locus. This structural alteration at the Gli-D2 locus may provide a genetic resource in breeding programs for the reduction of CD immunotoxicity.