An increased number of high molecular weight glutenin subunits (HMW-GSs) is advantageous to the formation of glutenin macropolymer, the major determinant of end-product quality. Out of the six HMW-GS genes, Glu-1Ay is typically inactive in most hexaploid wheat cultivars. In the current study, we characterized two active Glu-1Ay genes Glu-1Ay N11 and Glu-1Ay C422 from a hexaploid wheat cultivar N11 and a durum wheat cultivar C422, respectively. The molecular masses of the 1Ay subunit of N11 (Glu-1Ay N11 ) and C422 (Glu-1Ay C422 ) were 61.1 kDa and 63.9 kDa, respectively, measured by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Molecular characterization of Glu-1Ay N11 and Glu-1Ay C422 showed their complete coding regions were 1764 bp and 1827 bp in length, respectively. The deduced amino acid sequence of both Glu-1Ay N11 and Glu-1Ay C422 has a similar typical primary structure as the other y-type HMW-GSs, but in comparison to Glu-1By and Glu-1Dy subunits, the Glu-1Ay subunit has one less conserved cysteine residue in the repetitive domain. Phylogenetic analysis revealed that the inactive Glu-1Ay had a closer evolutionary relationship with Glu-1Ay C422 than with Glu-1Ay N11 . The divergence time among Glu-1Ay N11 , Glu-1Ay C422 , and other y-type subunit genes was also estimated.