Gliadin is a group of grain storage proteins that confers extensibility/viscosity to the dough and are vital to end-use quality in wheat. Moreover, gliadins are one of the important components for nutritional quality because they contain the nutritional unprofitable epitopes that cause chronic immune-mediated intestinal disorder in genetically susceptible individuals designated celiac disease (CD). The main genetic loci encoding the gliadins were revealed by previous studies; however, the genes related to the content of gliadins and their fractions were less elucidated. To illustrate the genetic basis of the content of gliadins and their fractions comprehensively, a recombinant inbred line (RIL) population that consisted of 196 lines was constructed from the two parents, Luozhen No.1 and Zhengyumai 9987. Quantitative trait loci (QTL) controlling the content of total gliadins and their fractions (ω-, α-, and γ-gliadin) were screened genome-widely under four environments across 2 years. Totally, thirty QTL which explained 1.97–12.83% of the phenotypic variation were detected to be distributed on 17 chromosomes and they were gathered into 12 clusters. One hundred and one pairs of epistatic QTL (E-QTL) were revealed, among which five were involved with the total gliadins and its fractions content QTL located on chromosome 1AS, 1DS, 4DS, 1DL, and 6AS. Three Kompetitive Allele-Specific PCR (KASP) markers were developed from three major QTL clusters located on chromosomes 6A, 6D, and 7D, respectively. The present research not only dissects the genetic loci for improving the content of gliadins and their three fractions, but may also contribute to marker-assisted selection of varieties with appropriate gliadin fractions content for end-use quality and health benefit at the early developmental stages and early breeding generations.