In this study, bicrystals of silver (Ag) and aluminum (Al) were used to investigate the physical spatial resolution of the electron backscatter diffraction system combining a digital image correlation method. Furthermore, the effect of the accelerating voltage and probe current was investigated on the physical spatial resolution of the lateral and longitudinal resolutions for Ag and Al, respectively. The lateral and longitudinal resolutions show high dependency on the accelerating voltage for a low atomic mass material of Al, In addition, these are almost independent of the accelerating voltage for a high atomic mass material of Ag. Moreover, the probe current does not play any role on both the lateral and longitudinal resolutions. The best lateral resolutions for Al and Ag are 40.5 and 12.1 nm at 10 kV and 1 nA, respectively. The best longitudinal resolutions of 23.2 and 80 nm were obtained at 10 kV and 1 nA for Al and Ag, respectively.