The present study examined the evolution of interfacial microstructure and on the element distribution of GH4169/GH3039 dissimilar joints by laser welding under different preheating temperatures. The results indicated that the microstructure observed in the interface layer on the GH4169 side exhibited distinct layers, namely diffusion layer and transition layer, which varied depending on the varied preheating temperature. Within the weld seam (WS), two types of dendrites were predominantly present: lath-like columnar dendrite and cellular dendrite. In the GH3039 side, the grain structure was mainly dominated by columnar dendrites, exhibits perpendicular growth to the fusion line and demonstrates a certain epitaxial growth morphology. Moreover, excessive preheating temperature intensified the migration of elements and phase transition behavior. As the preheating temperature increased, the low-angle grain boundaries (LAGBs) percentage decreased from 13% to 9.4%. The average grain size of the WS at room temperature, 200°C and 400°C is 44.1 μm, 53.3 μm and 58.2 μm, respectively. This paper provides a basis for tuning microstructure and element behavior via preheating temperature optimization in laser welding of dissimilar GH4169/GH3039 for further engineering application.