This work focuses on homoepitaxial growth of β-Ga2O3 on (100)-oriented substrates during molecular beam epitaxy. It provides a comprehensive study on the growth mode by combining in situ with ex situ tools. In situ reflection high-energy electron diffraction (RHEED) indicates 2D layer-by-layer mode accompanied by (1 × 1) surface reconstruction. The homoepitaxial layers are grown pseudomorphic with the substrate without in-plane strain as probed by in-plane azimuthal RHEED and out-of-plane synchrotron-based high resolution x-ray diffraction. In contrast to the substrate, stacking faults and twin domains are present in the layer.