Wheat (Triticum aestivum L., AABBDD) high molecular weight glutenin subunits (HMW-GS) were encoded by the genes located in Glu-A1, Glu-B1, and Glu-D1 loci. Evaluation and optimization of the combination of HMW-GS are very important to understand Glu-1 functions. In this study, we constructed a HMW-GS deletion mutant, DLGlu1 with Xiaoyan 81 background, and crossed it with Glenlea, a Canada elite wheat variety with superior end-use quality. Combining the technologies of wheat embryo culture and molecular marker-assisted selection (MAS), we obtained seven introgression lines containing Glenlea Glu-A1a, Glu-B1al, and Glu-D1d loci, which can be developed as a complete set of near-isogenic introgression lines possessing Glenlea different HMW-GS genes. Our study indicated that the Glu-1 deletion mutant DLGlu1 is of great value in the fast development of Glu-1 near-isogenic introgression lines and the study and utility of wheat Glu-1.