Flexible and water-insoluble regenerated silk materials have caught considerable interest due to their mechanical properties and numerous potential applications in medical fields. In this study, regenerated Mori (China), Thai, Eri, Muga, and Tussah silk films were prepared by a formic acid-calcium chloride (FA) method, and their structures, morphologies, and other physical properties were comparatively studied through Fourier transform infrared spectroscopy (FTIR), wide-angle X-ray scattering (WAXS), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), and thermogravimetric analysis (TGA). FTIR results demonstrated that the secondary structures of those five types of silk films are different from those of their respective natural silk fibers, whose structures are dominated by stacked rigid intermolecular β-sheet crystals. Instead, intramolecular β-sheet structures were found to dominate these silk films made by FA method, as confirmed by WAXS. We propose that silk I-like structures with intramolecular β-sheets lead to water insolubility and mechanical flexibility. This comparative study offers a new pathway to understanding the tunable properties of silk-based biomaterials.