The unique gluten characteristics of common wheat makes it suitable for the production of diverse food products. The composition and content of its two major components, glutenin and gliadin, are crucial factors influencing the properties of gluten protein and the end use quality of wheat. In this study, a gliadin gene, TaGli-γ-2.1, was identified associated with gluten strength through RNA-seq in a set of varieties with divergent gluten strength. Bisulfite sequencing revealed that the expression level of TaGli-γ-2.1 was negatively correlated with the cytosins methylation level in its promoter region. Hypermethylated TaGli-γ-2.1 conferred with decrease of γ-gliadin and total gliadin accumulation, accordingly, increase of gluten strength. Furthermore, 41 and 30 QTLs were screened for controlling the content of glutenin and gliadin fractions in a recombinant inbreed lines (RIL) population. These QTLs were detected in clusters. Seven and 12 clusters enriched with QTLs for more than three traits were identified for the content of glutenin and gliadin fractions. Finally, 5 Kompetitive Allele-Specific PCR markers were developed from the main QTL clusters and validated as significantly associated with the target traits in the RIL population and in natural varieties. On the basis of this study and the KASP markers described herein, breeders will be able to efficiently select wheat lines with favorable gluten properties at the early developmental stages and early breeding generations.