This study aimed to validate and implement a methodology in which fiducials implanted in the periphery of lung tumors can be used to reduce uncertainties in tumor location. Alignment software that matches marker positions on two-dimensional (2D) kilovoltage portal images to positions on three-dimensional (3D) computed tomography data sets was validated using static and moving phantoms. This software also was used to reduce uncertainties in tumor location in a patient with fiducials implanted in the periphery of a lung tumor. Alignment of fiducial locations in orthogonal projection images with corresponding fiducial locations in 3D data sets can position both static and moving phantoms with an accuracy of 1 mm. In a patient, alignment based on fiducial locations reduced systematic errors in the left-right direction by 3 mm and random errors by 2 mm, and random errors in the superior-inferior direction by 3 mm as measured by anterior-posterior cine images. Software that matches fiducial markers on 2D and 3D images is effective for aligning both static and moving fiducials before treatment and can be implemented to reduce patient setup uncertainties.