Titanium alloy Ti-6Al-4V processed by additive manufacturing (AM) has great potential for many high-performance applications including biomedical implants and aerospace components. However, typical microstructures obtained through AM are indeed very complex, and this often causes anisotropic properties. A systematic multi-scale microstructural analysis of AM parts is, thus, required to establish a correlation between deposition conditions, microstructures and properties. In the current work, electron beam melting (EBM) was used to 3D print Ti-6Al-4V builds using different scanning strategies. Through a detailed electron backscatter diffraction (EBSD) analysis, it was found that the microstructural characteristics, in particular alpha phase size, morphology and variant selection, change significantly either with a change in the EBM scanning pattern or across the build direction in each build. This is believed to arise primarily from the change in cooling rates and thermal gradients during the build process. A detailed analysis of the variant selection mechanism and intervariant boundaries character was carried out. This is a major step towards correlating the microstructural properties of electron beam melted Ti-6Al-4V alloy to the thermal history.