Post-irradiation annealing (PIA) behavior of irradiation-induced microstructural changes and hardening of two kind of A533B (first generation (1stGENS: 0.16 wt.% Cu) and second generation (2ndGENS: 0.04 wt.% Cu)) steels after neutron-irradiation of 3.9 × 1019 n cm-2 at 290 °C has been studied by positron annihilation spectroscopy, atom probe tomography and Vickers microhardness measurements. In the 1stGENS, clear two recovery stages are observed: (i) as-irradiated state to 450 °C and (ii) 450 to 600 °C. The first stage is due to annealing out of the most of irradiation-induced vacancy-related defects (VRDs), and the second stage corresponds to dissolving irradiation-induced Cu-rich solute nano-clusters (CRSCs). The experimental hardening is almost twice of the hardening due to the CRSCs estimated by Russell-Brown model below 350 °C, but almost the same as the estimation from 400 to 550 °C. In the 2ndGENS, the VRDs and non-Cu-rich solute nano-clusters (NCRSCs) recover at 450 °C. No CRSC has been formed even in all the annealing process. The experimental hardening is almost twice of the hardening estimated due to the NCRSCs by Russell-Brown model below 400 °C.