This study evaluates the difference in defining the target volume of rectal cancer with MRI-guided radiation treatment planning. Tumors show different appearances on different MR sequences, and also the motion of patients during the scan may affect defining planning target volume. A quantitative radial distance method was developed to evaluate variations coming from image contrast and patient motion. A total of 45 patients with pre- and post-radiation treatment MRI were analyzed. The mean difference in the radial distance between ROI’s drawn on different post-contrast images was 2-3 mm, and the difference in the 90th percentile tumor pixel was 6-8 mm.