This study characterizes an electron beam welded dissimilar joint between P91 ferritic/martensitic steel and Incoloy 800HT Nickel-based alloy. Such dissimilar metal welds (DMW) have applications in nuclear and thermal powerplants where steam-carrying pipes lead to header sections operating at around 600–650 °C. The study employed an advanced welding process to obtain a clean, high-quality dissimilar metal. The microstructure characterization of the DMW involving Nickel-based alloys is prone to solidification cracking in the weld fusion zone (WFZ), which affects the overall weld properties and leads to failure. In the present study, the WFZ microstructure revealed a mixed solidification mode with a homogenous presence of equiaxed dendritic and cellular-type structures. Advanced analyses of WFZ microstructure were done to account for microsegregation. Nb, Ti, Cr, and Mo microsegregation was found in the WFZ. The Ti-rich grain boundaries in the WFZ provided the pinning action during plastic deformation. The evolution of Cr, Mo-based carbide phases affected the microhardness distribution. Microsegregation under rapid solidification of the WFZ resulted in the formation of secondary phases in the interdendritic region. The effect of the evolution of precipitates deteriorated the mechanical strength. High and uneven dislocation distribution was observed in the WFZ near the IN 800HT weld interface. Under tensile loading, the DMW failed from the IN 800HT weld interface due to coarsening of precipitated in the IN 800HT HAZ region near the weld interface and grain boundary thickening due to the deposition of Ti over them. The tensile strength of WFZ was 592 MPa. The tensile strength of WFZ was 592 MPa. The post-weld heat treatment (PWHT) homogenized the microhardness across the weld. Impact toughness after PWHT slightly increased from 63 J to 69 J. The dissimilar joint obtained using the EBW process had sufficient mechanical strength to be considered for Generation IV power plants under the American Society for Testing and Materials (ASTM) standards. The study concludes that the dissimilar metal welded joint was highly prone to failure despite sufficient strength, as IN 800HT weld interface was susceptible to grain boundary liquation.