Common wheat (Triticum aestivum, 2n = 6x = 42, AABBDD) is the most widely cultivated staple food crop in the world. Wheat flours can be processed into diverse types of food products (e.g., bread, noodles, and biscuits) mainly due to the function of a unique group of seed proteins, i.e., gluten proteins, which include high-molecular-weight glutenin subunits (HMW-GSs), low-molecular-weight glutenin subunits (LMW-GSs), and gliadins. HMW-GSs and LMW-GSs are encoded by homoeologous Glu-1 and Glu-3 loci, respectively, with the former located on the long arms of 1A, 1B, or 1D chromosomes and the latter on the short arms of the same set of chromosomes. Gliadins are specified by either homoeologous Gli-1 (located on the short arm of 1A, 1B, or 1D) or Gli-2 loci (on the short arm of 6A, 6B, or 6D). HMW-GSs, LMW-GSs, and gliadins often show significant variations in both content and composition among different cultivars. This confers varying elastic and extension properties to the doughs prepared from different wheat flours, thus giving rise to different end-use qualities. However, gluten proteins, especially gliadins, carry the epitopes that cause celiac disease or a variety of other gluten sensitivities in susceptible human individuals. Collectively, these findings have placed gluten proteins as the central players in shaping and improving wheat end-use and health-related qualities. Therefore, we have conducted a systematic mutagenesis analysis of gluten protein genes and loci using the winter wheat varieties Xiaoyan 81 and Xiaoyan 54, with the resulting mutants used in understanding and improving gluten protein functions in end-use quality control. By screening ion-beam mutant population of Xiaoyan 81 and conducting dedicated crossings between primary mutants, we have developed a series of mutants lacking one, two, or all three Glu-1 loci, a second series of mutants lacking one, two, or all three Gli-1/Glu-3 compound loci, a third set of mutants lacking various combinations of Glu-1 and Gli-1/Glu-3 compound loci, a fourth set of mutants devoid of one or two Gli-2 loci, and a fifth set of mutants devoid of all gluten protein loci carried by subgenome A, with the six set of mutants lacking all gluten protein loci carried by subgenome D coming soon. In the mean time, multiple knockout and missense EMS mutants were developed for the five HMW-GSs of Xiaoyan 54. Furthermore, we have demonstrated the values of our gluten protein mutants in understanding and improving wheat breadmaking and nutritional qualities, and developed a commercial strong gluten wheat cultivar, i.e., Kexing 3302, using the elite 1Ax1G330E mutant allele. We will continue the efforts to generate and analyze gluten protein mutants in the future, thus making more and innovative contributions to the breeding of high-quality wheat with valuable health-promoting attributes.