AbstractThe self‐assembly of anisotropic nanocrystals (stabilized by organic capping molecules) with pre‐selected composition, size, and shape allows for the creation of nanostructured materials with unique structures and features. For such a material, the shape and packing of the individual nanoparticles play an important role. This work presents a synthesis procedure for ω‐thiol‐terminated polystyrene (PS‐SH) functionalized gold nanooctahedra of variable size (edge length 37, 46, 58, and 72 nm). The impact of polymer chain length (Mw: 11k, 22k, 43k, and 66k g⋅mol⁻¹) on the growth of colloidal crystals (e. g. mesocrystals) and their resulting crystal structure is investigated. Small‐angle X‐ray scattering (SAXS) and scanning transmission electron microscopy (STEM) methods provide a detailed structural examination of the self‐assembled faceted mesocrystals based on octahedral gold nanoparticles of different size and surface functionalization. Three‐dimensional angular X‐ray cross‐correlation analysis (AXCCA) enables high‐precision determination of the superlattice structure and relative orientation of nanoparticles in mesocrystals. This approach allows us to perform non‐destructive characterization of mesocrystalline materials and reveals their structure with resolution down to the nanometer scale.