OBJECTIVE The purpose of this study was to evaluate regional differences of canine stifle articular cartilage using the quantitative magnetic resonance imaging (MRI) technique of T2 mapping. METHODS Fourteen stifle joints from seven juvenile male Beagle dogs with no evidence or prior history of pelvic limb lameness were imaged ex vivo using standard of care fast spin echo MRI and quantitative T2 mapping protocols. Regions of interest were compared between the femoral, patellar and tibial cartilages, as well as between the lateral and medial femorotibial compartments. Limbs were processed for histology with standard stains to confirm normal cartilage. RESULTS The average T2 value of femoral trochlear cartilage (37.5 ± 2.3 ms) was significantly prolonged (p < 0.0001) as compared with the femoral condylar, patellar and tibial condylar cartilages (33.1 ± 1.5 ms, 32.8 ± 2.3 ms, and 28.0 ± 1.7 ms, respectively). When comparing medial and lateral condylar compartments, the lateral femoral condylar cartilage had the longest T2 values (34.8 ± 2.8 ms), as compared with the medial femoral condylar cartilage (30.9 ± 1.9 ms) and lateral tibial cartilage (29.1 ± 2.3 ms), while the medial tibial cartilage had the shortest T2 values (26.7 ± 2.4 ms). CONCLUSIONS As seen in other species, regional differences in T2 values of the canine stifle joint are identified. Understanding normal regions of anticipated prolongation in different joint compartments is needed when using quantitative imaging in models of canine osteoarthritis.