We have investigated a fully automatic setup error estimation method that aligns DRRs (digitally reconstructed radiographs) from a three-dimensional planning computed tomography image onto two-dimensional radiographs that are acquired in a treatment room. We have chosen a MI (mutual information)-based image registration method, hoping for robustness to intensity differences between the DRRs and the radiographs. The MI-based estimator is fully automatic since it is based on the image intensity values without segmentation. Using 10 repeated scans of an anthropomorphic chest phantom in one position and two single scans in two different positions, we evaluated the performance of the proposed method and a correlation-based method against the setup error determined by fiducial marker-based method. The mean differences between the proposed method and the fiducial marker-based method were smaller than 1 mm for translational parameters and 0.8 degree for rotational parameters. The standard deviations of estimates from the proposed method due to detector noise were smaller than 0.3 mm and 0.07 degree for the translational parameters and rotational parameters, respectively.