Relationships between microstructures and hardening nature of laser powder bed fused (L-PBF) 316 L stainless steel have been studied. Using integrated experimental efforts and calculations, the evolution of microstructure entities such as dislocation density, organization, cellular structure and recrystallization behaviors were characterized as a function of heat treatments. Furthermore, the evolution of dislocation-type, namely the geometrically necessary dislocations (GNDs) and statistically stored dislocations (SSDs), and their impacts on the hardness variation during annealing treatments for L-PBF alloy were experimentally investigated. The GND and SSD densities were statistically measured utilizing the Hough-based EBSD method and Taylor's hardening model. With the progress of recovery, the GNDs migrate from cellular walls to more energetically-favourable regions, resulting in the higher concentration of GNDs along subgrain boundaries. The SSD density decreases faster than the GND density during heat treatments, because the SSD density is more sensitive to the release of thermal distortions formed in printing. In all annealing conditions, the dislocations contribute to more than 50% of the hardness, and over 85.8% of the total dislocations are GNDs, while changes of other strengthening mechanism contributions are negligible, which draws a conclusion that the hardness of the present L-PBF alloy is governed predominantly by GNDs.