Images acquired from an electronic portal imaging device are aligned with digitally reconstructed radiographs (DRRs) or other portal images to verify patient positioning during radiation therapy. Most of the currently available computer aided registration methods are based on the manual placement of corresponding landmarks. The purpose of the paper is twofold: (a) the establishment of a methodology for patient set-up verification during radiotherapy based on the registration of electronic portal images, and (b) the evaluation of the proposed methodology in a clinical environment. The estimation of set-up errors, using the proposed methodology, can be accomplished by matching the portal image of the current fraction of the treatment with the portal image of the baseline treatment (reference portal image) using a nearly automated technique. The proposed registration method is tested on a number of phantom data as well as on data from four patients. The phantom data included portal images that corresponded to various positions of the phantom on the treatment couch. For each patient, a set of 30 portal images was used. For the phantom data (for both transverse and lateral portal images), the maximum absolute deviations of the translational shifts were within 1.5 mm, whereas the in-plane rotation angle error was less than 0.5 degrees. The two-way Anova revealed no statistical significant variability both within observer and between-observer measurements (P > 0.05). For the patient data, the mean values obtained with manual and the proposed registration methods were within 0.5 mm. In conclusion, the proposed registration method has been incorporated within a system, called ESTERR-PRO. Its image registration capability achieves high accuracy and both intra- and inter-user reproducibility. The system is fully operational within the Radiotherapy Department of 'HYGEIA' Hospital in Athens and it could be easily installed in any other clinical environment since it requires standardized hardware specifications and minimal human intervention.