Electron beam powder bed fusion (EB-PBF) is a process to additively manufacture Ti–6Al–4V. Along with process induced defects, the unique microstructure of EB-PBF Ti–6Al–4V leads to anisotropic properties, and therefore an understanding of how the microstructure evolves as layers are added is required. This experimental study presents the microstructural evolution of Ti–6Al–4V during the deposition of initial layers in EB-PBF process. Two geometries of specimens, ramp and step, were fabricated from 0.1 mm thickness (2 layers) to 0.7 mm (14 layers), and 0.2 mm (4 layers) to 1.4 mm (28 layers). The evolution of the microstructure was investigated through optical and electron microscopy, spatial microhardness mapping, and detailed microstructural characterization. Up to the deposition of seven layers, the microstructure comprised of α+β phases with partially retained α′ phase from rapid solidification cooling that resulted in higher microhardness. A unique microstructural gradient composed of three distinct regions (Region I, II, III) appears along the build direction from the deposition of 10 layers until 28 layers. Region I showed a finer α+β microstructure in irregular prior β grain with partial martensitic α’ phase and displayed greater microhardness. Region II showed coarser α+β microstructure and lower microhardness than both Region I and III. The average α-lath thickness and α-volume fraction increased as new layers were deposited. Perpendicular to the build direction, the hardness distribution was observed to be uniform along the longitudinal x-y direction.