Electron backscatter diffraction (EBSD) analysis is applied to [0 0 1] oriented magnetite thin films grown on MgO substrates. A high image quality of the Kikuchi patterns was achieved enabling multi-phase scans. Several types of magnetite thin films were analyzed; one as-grown and the others after different annealing steps in oxygen atmosphere. From the EBSD mappings, we learn that the optimum orientation in [0 0 1]-direction is not yet achieved for the as-grown sample, but develops upon oxygen treatment. Furthermore, the distribution of misorientation angles within the investigated area (=1 grain) is found to change during the annealing steps. After 3 min of annealing, most of the misorientations around 30°–40° have vanished, and some islands with high misorientation angles remain, which may play a role as antiferromagnetic pinning centers.