The purpose of the paper is to present and evaluate the performance of a new softwarebased registration system for patient set-up verification, during radiotherapy, using electronic portal images. The estimation of set-up errors, using the proposed system, can be accomplished by means of registering the portal image of the current fraction of the treatment with the portal image of the first image of the treatment (reference portal image) by applying a nearly automated technique. The proposed system was tested on data obtained by a phantom 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 thirty portal images were used. The accuracy of the system was (in average) better than 1 mm and the estimated set-up error was very close to the expected one.