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The high molecular weight glutenin subunit (HMW-GS), encoded by the 1Ay gene, unexpressed in common wheat, exists in diploid and tetraploid wheats. An active 1Ay gene was first cloned from wild emmer wheat (T. turgidum ssp. dicoccoides, 2n = 4x = 28, AABB), the oldest species in emmer wheat. Here, a novel subunit encoded by the 1Ay gene (JF519636) present in T. turgidum ssp. dicoccoides line D141 was characterized. The protein had 608 amino acids with six cysteine residues and showed faster electrophoretic mobility than 1Dy12 from common wheat. Compared with previously reported 1Ay subunits, it contained 16 single point mutations (SPMs). Comparative and phylogenetic sequence analyses suggested that this gene was more similar to the 1Ay gene from the diploid species (2n = 2x = 14, AA) T. urartu than from T. monococcum ssp. aegilopoides. Its predicted secondary protein structure possessed a different content of motifs relative to the 1Ay gene (AY245578) from T. urartu, which had similar electrophoretic mobility. In the central repetitive domain, JF519636 had more β-turns and β-bends than the 1Ay subunit AY245578. These structural characteristics in JF519636 could possibly be associated with specific gluten properties.