Objective Magnetic resonance imaging is the standard imaging modality to assess articular cartilage. As the imaging surrogate of degenerative joint disease, cartilage thickness is commonly quantified after tissue segmentation. In lack of a standard method, this study systematically compared five methods for automatic cartilage thickness measurements across the knee joint and as a function of region and sub-region: 3D mesh normals (3D-MN), 3D nearest neighbors (3D-NN), 3D ray tracing (3D-RT), 2D centerline normals (2D-CN), and 2D surface normals (2D-SN). Design Based on the manually segmented femoral and tibial cartilage of 507 human knee joints, mean cartilage thickness was computed for the entire femorotibial joint, 4 joint regions, and 20 subregions using these methods. Inter-method comparisons of mean cartilage thickness and computation times were performed by one-way analysis of variance (ANOVA), Bland-Altman analyses and Lin’s concordance correlation coefficient (CCC). Results Mean inter-method differences in cartilage thickness were significant in nearly all subregions ( P < 0.001). By trend, mean differences were smallest between 3D-MN and 2D-SN in most (sub)regions, which is also reflected by highest quantitative inter-method agreement and CCCs. 3D-RT was prone to severe overestimation of up to 2.5 mm. 3D-MN, 3D-NN, and 2D-SN required mean processing times of ≤5.3 s per joint and were thus similarly efficient, whereas the time demand of 2D-CN and 3D-RT was much larger at 133 ± 29 and 351 ± 10 s per joint ( P < 0.001). Conclusions In automatic cartilage thickness determination, quantification accuracy and computational burden are largely affected by the underlying method. Mesh and surface normals or nearest neighbor searches should be used because they accurately capture variable geometries while being time-efficient.