Different methods have been used to cross-validate cartilage thickness measurements from magnetic resonance images (MRIs); however, a majority of these methods rely on interpolated data points, regional mean and/or maximal thickness, or surface mean thickness for data analysis. Furthermore, the accuracy of MRI cartilage thickness measurements from commercially available software packages has not necessarily been validated and may lead to an under- or overestimation of cartilage thickness. The goal of this study was to perform a matching point-to-point validation of indirect cartilage thickness calculations using a magnetic resonance (MR) image data set with direct cartilage thickness measurements using biomechanical indentation testing at the same anatomical locations. Seven bovine distal femoral condyles were prepared and a novel phantom filled with dilute gadolinium solution was rigidly attached to each specimen. High resolution MR images were acquired, and thickness indentation analysis of the cartilage was performed immediately after scanning. Segmentation of the MR data and cartilage thickness calculation was performed using semi-automated software. Registration of MR and indentation data was performed using the fluid filled phantom. The inter- and intra-examiner differences of the measurements were also determined. A total of 105 paired MRI-indentation thickness data points were analyzed, and a significant correlation between them was found (, ). The mean difference (±std. dev.) between measurement techniques was , with Bland–Altman limits of agreement of 0.45 mm and −0.46 mm. The intra- and inter-examiner measurement differences were and , respectively. This study validated cartilage thickness measurements from MR images with thickness measurements from indentation by using a novel phantom to register the image-based and laboratory-based data sets. The accuracy of the measurements was comparable to previous cartilage thickness validation studies in literature. The results of this study will aid in validating a tool for clinical evaluation of in-vivo cartilage thickness.