Low-molecular-weight glutenin subunits (LMW-GSs) are one of the important factors for the dough processing quality. In this study, a novel LMW-GS, designated LMW-N13, from the wheat relative species Aegilops uniaristata PI 554421 was cloned and characterized. Unlike previously published LMW-GSs, LMW-N13 has a large molecular weight and is the largest LMW-GS published thus far. Sequence alignments demonstrated that LMW-N13 is a LMW-i-type subunit but contains nine cysteine residues which is one more than typical LMW-i-type subunits. In addition, four insertions are present in the repetitive domain that resulted in the large molecular weight. In vitro analysis showed that LMW-N13 could improve the dough quality of different base flours.