Butter oil is derived from butter and constitutes of triglycerides along with small amounts of other lipids and fat-soluble components. Food grade lipids can be easily sourced from butter oil which finds a great potential in various ancient, modern as well as emerging applications. Due to almost “all fat” content, butter oil is not soluble in water, but it can be emulsified or combined with other components to enhance its applicability. In order to develop and optimize various applications, it is vital to identify self-assembled nanostructures formed within the butter oil. This report involves nanostructural studies by small (SAXS) and wide (WAXS) angle X-ray scattering and microstructural analysis of butter and butter oil using microscopic techniques. Both butter and butter oil display various polymorphs, detected by WAXS, but in general, the self-assembled nanostructure was identified to be a lamellar phase with the lattice parameter of about 41.8 Å. Physicochemical properties of butter oil, namely solubility, density, thermal behavior, functional groups and molecular structure elucidation also contribute to this report.