The objective of this work was to develop a methodology for measuring cartilage thickness in anatomically based subregions in the tibial and in the central weight-bearing femoral cartilage from magnetic resonance (MR) images. The tibial plateau was divided into a central area of the total subchondral bone area (tAB), and anterior, posterior, internal, and external subregions surrounding it. In the weight-bearing femoral condyles, central, internal, and external subregions were determined. The Euclidean distance between the tAB and cartilage surface was used for determining cartilage thickness. The reproducibility of the method was evaluated on test-retest data sets of 12 participants (six healthy, six with osteoarthritis). The subregion size was varied systematically to study the influence on the reproducibility. The size of the subregions was highly consistent under conditions of repositioning (standard deviation 0.0%-0.3%). The precision errors for regional mean cartilage thickness measurements ranged from 19 microm (1.5%) to 84 microm (4.7%). The computation of regional cartilage thickness values from segmented MR images is shown to be highly reproducible and robust under conditions of joint repositioning. In longitudinal studies, this technique may substantially enhance the ability of quantitative MRI to monitor structural changes in osteoarthritis at narrow time intervals.