The high-molecular-weight glutenin subunits (HMW-GS) represent a major component of the endosperm storage protein in the grains of wheat and its related species. Their technological importance results from their ready formation of intermolecular disulfide bonds, which underlie much of the visco-elasticity displayed by gluten and hence the processing quality of the flour. Here, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the Chinese wheat cultivar Xiaoyanmai 7 formed four distinct HMW-GS, two of which are likely the product of a known allele at the Glu-B1 locus, whereas the other two did not match any known HMW-GS. A combined analysis based on reversed-phase high-performance liquid chromatography (RP-HPLC), N-terminal sequencing and mass spectrometry confirmed that the two novel proteins were genuine HMW-GS. Inspection of the DNA sequences showed that one of the novel HMW-GS was encoded by an x-type and the other by a y-type secalin gene. A karyotypic analysis confirmed that six of the seven pairs of Xiaoyanmai 7’s D genome chromosomes (the exception was chromosome 2D) had been replaced by rye chromosomes. The y-type HMW secalin present in Xiaoyanmai 7 differed from the standard By and Dy HWM-GS by the presence of an additional cysteine residue in its C-terminal domain.