A wheat mutant of low-molecular-weight glutenin (LMW-GS) “Gunji-2” at Glu-B3 locus was derived among the double haploid lines. Gunji-2 was derived from F1 plants of Keumkang and Olgeuru crosses using the wheat × maize system according to the procedures of Inagaki and Mujeeb-Kazi at International Maize and Wheat Improvement Center (CIMMYT). Deletion of Glu-B3 LMW-GS proteins was found by allele specific DNA marker, one dimensional SDS-PAGE and two dimensional gel electrophoresis (2-DGE). Tandom mass spectrometry (MS/MS) was used to obtain direct evidence of LMW-GS deletion. In addition, we examined the basic agronomic traits, protein content, dough properties of mixing and bread loaf volumeof Gunji-2 and parental wheat cultivars grown for two years. This mutant will represent a valuable resource in quality test for specific allele or gene at Glu-B3 locus.