The microstructure of additively manufactured 316L stainless steel is hierarchical, and on a fine scale, it contains cell structures and dislocations. These microstructures define the mechanical properties, and it is thus of importance to quantify them and understand their thermal stability. This study investigates the heterogeneity of the microstructure in laser powder bed-fused 316L with a focus on variations in the cell and dislocation structures through the sample thickness along the build direction. While at the coarse scale the microstructure is rather homogeneous throughout its thickness, there are significant variations in the dislocation network, highlighting a higher dislocation density near the bottom layers than near the top. Furthermore, post-processing heat treatment at 500 °C and 800 °C reveals different stabilities of the cell structures, with significant cell dissolution at 800 °C, particularly at the top of the build. Microhardness measurements corroborate these findings, showing higher hardness in the bottom layers across all conditions, e.g., an increase in hardness from 225 HV to 236 HV is observed in the as-built condition. These results underpin the suggestion that significant microstructural heterogeneity may exist through the thickness in as-built parts, which affects the mechanical properties and subsequent heat treatments.