Seven Glu-A1m allelic variants of the Glu-A1mx genes in Triticum monococcum ssp. monococcum, designated as 1Ax2.1a, 1Ax2.1b, 1Ax2.1c, 1Ax2.1d, 1Ax2.1e, 1Ax2.1f, and 1Ax2.1g were characterized. Their authenticity was confirmed by successful expression of the coding regions in E. coli, and except for the 1Ax2.1a with the presence of internal stop codons at position of 313 aa, all correspond to the subunit in seeds. However, all the active six genes had a same DNA size although their encoding subunits showed different molecular weight. Our study indicated that amino acid residue substitutions rather than previously frequently reported insertions/deletions played an important role on the subunit evolution of these Glu-A1mx alleles. Since variation in the Glu-A1x locus in common wheat is rare, these novel genes at the Glu-A1mx can be used as candidate genes for further wheat quality improvement.