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Contrast‐enhanced computed tomography (CECT) using charged contrast‐agents enables quantification of cartilage glycosaminoglycan content. Since glycosaminoglycan content is a key determinant of cartilage compressive stiffness, CECT measurements have the potential to non‐invasively assess cartilage stiffness. The objective of this study was to determine whether CECT attenuation, using a cationic contrast‐agent (CA4+), correlates with the stiffness of intact cartilage. Six fresh femoral and six fresh tibial compartments with intact cartilage were obtained from patients undergoing total knee replacement surgery. The instantaneous stiffness was determined for 25–50 points on the surface of each compartment using an established indentation technique. The samples were then immersed in CA4+ solution for 48 h, scanned in a micro‐CT scanner, and the average CECT attenuation at each indentation site was found for the superficial cartilage. A significant (p < 0.01) and positive correlation was observed between stiffness and CECT attenuation for sites from each individual cartilage surface, with correlation coefficients ranging from r = 0.37–0.57 and r = 0.48–0.69 (p < 0.01) for the tibia and femur, respectively. When data for each type of cartilage surface were pooled together, the correlation coefficients were r = 0.73 for femoral condyle data points and r = 0.49 for tibial plateau data points. CECT provided a map of cartilage stiffness across each surface, which allows regions of low stiffness to be identified. These findings support continued evaluation and development of quantitative imaging techniques to assess the functional properties of cartilage. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2641–2647, 2018. Contrast‐enhanced computed tomography (CECT) using charged contrast‐agents enables quantification of cartilage glycosaminoglycan content. Since glycosaminoglycan content is a key determinant of cartilage compressive stiffness, CECT measurements have the potential to non‐invasively assess cartilage stiffness. Therefore, we measured stiffness in intact human knee cartilage using indentation and measured cartilage glycosaminoglycan content by CECT imaging. We found a statistically significant and positive correlation between indentation and CECT measurements. CECT provided a map of cartilage stiffness across each surface, which allows regions of low stiffness to be identified. These findings support continued evaluation and development of quantitative imaging techniques to assess the functional properties of cartilage. [ABSTRACT FROM AUTHOR]