Some recent studies have revealed individual and the combined interactions of gluten and starch affecting dough mixing properties. However, the combined influence of high-molecular-weight glutenin subunits (HMW-GS) and starch on dough mixing and rheological properties requires elucidation. Thus four recombinant inbred lines, SS 1, SS 2, ZZ 1 and ZZ 2, were selected based on their HMW-GSs compositions. Compared to ZZ 1 and ZZ 2, both SS 1 and SS 2 carried superior HMW-GS alleles, and exhibited extended dough development and stability time, indicating their significant dough mixing characteristics. The gluten skeleton of the wheat lines SS 2 and ZZ 2 with higher B-type starch proportions exhibited fewer breakages along with the rise of dough temperature during mixing. Higher content of B-type starch strengthens interaction between starch and gluten skeleton at the dough heating stage, suggesting a specific range of B-type starch proportion can improve dough mixing characteristics.