Cellulose is crystallized by plants and other organisms into fibrous nanocrystals. The mechanical properties of these nanofibers and the formation of helical superstructures with energy dissipating and adaptive optical properties depend on the ordering of polysaccharide chains within these nanocrystals, which is typically measured in bulk average. Direct measurement of the local polysaccharide chain arrangement has been elusive. In this study, we use the emerging technique of scanning electron diffraction to probe the packing of polysaccharide chains across cellulose nanofibers and to reveal local ordering of the chains in twisting sections of the nanofibers. We then use atomic force microscopy to shed light on the size dependence of the inherent driving force for cellulose nanofiber twisting. The direct measurement of crystalline twisted regions in cellulose nanofibers has important implications for understanding single-cellulose-fibril properties that influence the interactions between cellulose nanocrystals in dense assemblies. This understanding may enable cellulose extraction and separation processes to be tailored and optimized.