In common wheat (Triticum aestivum L.) and its relative species, considerable progress has been made in understanding the structure and function of the high-molecular-weight glutenin subunit (HMW-GS). As a species closely related to wheat, Aegilops umbellulata is an important resource for wheat genetic improvement. In this paper, we report a novel HMW-GS 1Ux3.5 in Aegilops umbellulata Y361. The complete open reading frame (ORF) coding for 1Ux3.5 was cloned and sequenced. Analysis of the deduced amino acid sequence revealed that the primary structure of 1Ux3.5 was similar to those of previously published HMW-GSs. The 1Ux3.5 possessed an extra cysteine residue in the repetitive domain, indicating that the subunit may be related to excellent dough quality. Subsequently, the single proteins of 1Ux3.5 and 1Dx5 (used as positive control) were purified at a scale sufficient for incorporation into flour for a dough quality test. Both the SDS sedimentation volume and mixograph parameters demonstrated that 1Ux3.5 showed a greater contribution to the dough quality than 1Dx5. Therefore, the 1Ux3.5 subunit from Aegilops umbellulata may have potential value in improving the processing properties of hexaploid wheat varieties.