The importance of comprehensive multiscale characterisation in advancing our understanding of engineering materials is undeniable but remains a challenging pursuit. Combining complimentary microstructure characterisation techniques, including transmission electron microscopy, electron backscatter diffraction and dark-field X-ray microscopy (DFXM), the formation of deformation microstructures is investigated in presence of shearable and non-shearable hardening precipitates in an industrial aluminium alloy (AA) 2024 (Al–Cu–Mg family). The alloy was used in naturally aged T3 (with shearable co-clusters and Guinier–Preston–Bagaryatsky (GPB) zones) and peak-hardened T8 (with non-shearable S-phase precipitates) states. After cold rolling with thickness reductions varying from 25 to 60% (or corresponding von Mises strain from 0.33 to 1.06), the T8 state revealed a higher sub-boundary density with slightly smaller mean disorientation angle, as compared to those in the T3 state. At a von Mises strain of 0.33, the T8 state exhibited higher long-range orientation gradients, as compared to the T3 state, for higher strain orientation gradients in T3 surpass those in T8 state. With DFXM, distinct 3D substructures are shown, revealing ellipsoidal sub-grains in the T8 state and pancake-like sub-grains in the T3 state. Moreover, the stored energy induced by cold rolling is higher for the T8 state. These results indicate different deformation microstructures, formed in the same AA2024 but hardened by shearable and non-shearable precipitates.