Prolamins extracted from wheat, rye and barley cultivars were analysed for colour characteristics, SDS-PAGE, Amino Acid, Dynamic Light Scattering (DLS), Zeta Potential, Scanning Electron Microscopy (SEM), Energy Dispersive X-Ray (EDX), Transmission Electron Microscopy (TEM) and X-ray Diffraction (XRD) to elucidate the structure. Amino acid analysis showed significant variation among the prolamins and the predominant essential amino acids were found to be leucine, phenylalanine and valine whereas predominant non-essential amino acids were glutamic acid, arginine and aspartic acid. All the prolamins exhibited positive zeta-potential, however gliadin had a significantly higher zeta potential value. TEM studies of prolamins revealed the compact globular structure but gliadin also had rod-shaped structure. Morphology by SEM illustrated the globular particle arrangement in gliadin and sheet like arrangement in secalin and hordein. XRD pattern of prolamin showed the ordered crystalline domain of prolamin at 44.1°, 37.8° and 10.4°. The d-spacing obtained from XRD and TEM analysis also supports the crystalline domain of prolamin apart from amorphous domain.