The microstructural evolutions and variations in mechanical performance of electron beam welded (EBW) Ti-6Al-4V (Ti64) alloy have been investigated. The effects of heat treatment on the microstructure of welded samples have been studied after post-welding solution treatment and ageing. The martensitic phase α′\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\alpha }{\\prime}$$\\end{document} has been confirmed using transmission electron microscopy (TEM). Electron backscatter diffraction (EBSD) has been used to investigate the phase and grain morphology. Results showed that the martensitic α′ phase coarsened, the size of heat-affected zone (HAZ) changed and grains in the base materials (BMs) had grown after the post-weld heat treatments (PWHT). The tensile behaviour of electron beam welded Ti64 has been investigated using in situ tensile testing monitored by optical microscopy. The deformation and failure were directly revealed during the in situ tensile process. Results showed that the EBW Ti64 samples have different failure locations after receiving different post-weld heat treatments. The relationship between the post-weld heat treatments, microstructural evolution and mechanical properties of EBW Ti64 were investigated. Thermodynamic databases were used to predict mechanical properties—including the yield strengths—of the titanium alloy for different grain sizes, representing different post-weld heat treatment operations, and these were embedded into a finite element modelling framework to simulate the tensile testing specimens to understand the mechanical fields experienced such as stresses and strains, just prior to failure.