|Abstract Background Imaging cannot be performed during natural weightbearing in biomechanical studies using conventional closed-bore MRI, which has necessitated simulating weightbearing load on the joint. Upright, open MRI (UO-MRI) allows for joint imaging during natural weightbearing and may have the potential to better characterize the biomechanical effect of tibiofemoral pathology involving soft tissues. However open MRI scanners have lower field strengths than closed-bore scanners, which limits the image quality that can be obtained. Thus, there is a need to establish the reliability of measurements in upright weightbearing postures obtained using UO-MRI. Methods Knees of five participants with prior anterior cruciate ligament (ACL) rupture were scanned standing in a 0.5 T upright open MRI scanner using a 3D DESS sequence. Manual segmentation of cartilage regions in contact was performed and centroids of these contact areas were automatically determined for the medial and lateral tibiofemoral compartments. Inter-rater, test-retest, and intra-rater reliability were determined and quantified using intra-class correlation (ICC3,1), standard error of measurement (SEM), and smallest detectable change with 95% confidence (SDC95). Accuracy was assessed by using a high-resolution 7 T MRI as a reference. Results Contact area and centroid location reliability (inter-rater, test-retest, and intra-rater) for sagittal scans in the medial compartment had ICC3,1 values from 0.95–0.99 and 0.98–0.99 respectively. In the lateral compartment, contact area and centroid location reliability ICC3,1 values ranged from 0.83–0.91 and 0.95–1.00 respectively. The smallest detectable change in contact area was 1.28% in the medial compartment and 0.95% in the lateral compartment. Contact area and centroid location reliability for coronal scans in the medial compartment had ICC3,1 values from 0.90–0.98 and 0.98–1.00 respectively, and in the lateral compartment ICC3,1 ranged from 0.76–0.94 and 0.93–1.00 respectively. The smallest detectable change in contact area was 0.65% in the medial compartment and 1.41% in the lateral compartment. Contact area was accurate to within a mean absolute error of 11.0 mm2. Conclusions Knee contact area and contact centroid location can be assessed in upright weightbearing MRI with good to excellent reliability. The lower field strength used in upright, weightbearing MRI does not compromise the reliability of tibiofemoral contact area and centroid location measures.