The effect of high-molecular weight glutenin subunits (HMW-GSs) on gluten polymerization during biscuit making was investigated using a set of HMW-GS deletion lines. Results showed that the deletion of HMW-GSs improved the biscuit quality compared with the wild type (WT), especially in x-type HMW-GS deletion lines. Slight gluten depolymerization was observed during dough mixing, while progressive gluten polymerization occurred during biscuit baking. The deletion of HMW-GSs suppressed the polymerization of glutenin and gliadin compared with the WT during biscuit baking, especially in x-type HMW-GS deletion lines. These actions resulted in less elevation of the intermolecular β-sheet and ordered α-helix and altering the disulfide (SS) conformation to a less stable conformation in HMW-GS deletion lines compared with the WT during baking. Molecular dynamics simulation analysis further demonstrated that x-type HMW-GSs had higher thermal stability compared with y-type HMW-GSs during heating.