The correlation of microstructure evolution and hardening was studied in two kinds of A533B-1 steel with high and low levels of Cu irradiated in a range of dose from 0.32 to 9.9 × 10 19 n cm −2 ( E > 1 MeV) under a high flux of about 1.7 × 10 13 n cm −2 s −1 using three-dimensional local electrode atom probe (3DAP), positron annihilation (PA) techniques, and Vickers microhardness. The early rapid hardening was found to be caused by mainly matrix defects such as mono- or di-vacancies ( V 1 − V 2) and/or dislocations indicated by the PA analysis. The 3DAP analysis showed that dense dispersion of dilute Cu rich clusters and lean distribution of Mn–Ni–Si rich clusters, which were identified to possess the same dislocation-pinning effect by applying a Russell and Brown model, were responsible for large and small hardening in high- and low-Cu steels irradiated above 0.59 × 10 19 n cm 2, respectively.