Despite the increasing production of wheat gluten (WG) for industrial use, minor attention has been given to the impact of the separation procedure on the gluten quality. The purpose of the present study was to probe the effect of the separation treatments (harsh vs mild) on gluten structure, morphology, and performance in bio-based films. The harshly separated industrial WG showed aggregated and pre-cross linked structure in the starting material most likely due to shear forces during gluten separation from flour and heat effect during the drying procedures. Further, when the harshly separated WG was processed into films the pre-crosslinked starting material restricted new crosslinks formation and structural rearrangements at nano-scale. The mechanical integrity of the film was also affected resulting in films with low Young's modulus and strength. WG (from cultivars Diskette, Puntari, and Sleipner) recovered from mild separation showed relatively "native" non-destructed crosslinking pattern and not previously observed structural morphology at nano-scale. When processed into films the mildly separated WG showed well polymerized intimately crosslinked proteins both with disulfide and other covalent crosslinks. The nano-scale morphology showed lamellar and hexagonal arrangements, not reported so far in any study. The structural rearrangements among films from mildly separated WG resulted in materials with improved mechanical integrity as compared to films from harshly separated WG. The present study showed that the quality of WG is significantly affected by the separation procedure which also affects protein polymerization, nano-scale morphology, and tensile properties of films.