High‐resolution peripheral quantitative computed tomography (HR‐pQCT) is a newly developed in vivo clinical imaging modality. It can assess the 3D microstructure of cortical and trabecular bone at the distal radius and tibia and is suitable as an input for microstructural finite element (µFE) analysis to evaluate bone's mechanical competence. In order for microstructural and image‐based µFE analyses to become standard clinical tools, validation with a current gold standard, namely, high‐resolution micro‐computed tomography (µCT), is required. Microstructural measurements of 19 human cadaveric distal tibiae were performed for the registered HR‐pQCT and µCT images, respectively. Next, whole bone stiffness, trabecular bone stiffness, and elastic moduli of cubic subvolumes of trabecular bone in both HR‐pQCT and µCT images were determined by µFE analysis. The standard HR‐pQCT patient protocol measurements, derived bone volume fraction (BV/TV^d), trabecular number (Tb.N*), trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp), and cortical thickness (Ct.Th), as well as the voxel‐based direct measurements, BV/TV, Tb.N*, Tb.Th*, Tb.Sp*, Ct.Th, bone surface‐to‐volume ratio (BS/BV), structure model index (SMI), and connectivity density (Conn.D), correlated well with their respective gold standards, and both contributed to µFE‐predicted mechanical properties in either single or multiple linear regressions. The mechanical measurements, although overestimated by HR‐pQCT, correlated highly with their gold standards. Moreover, elastic moduli of cubic subvolumes of trabecular bone predicted whole bone or trabecular bone stiffness in distal tibia. We conclude that microstructural measurements and mechanical parameters of distal tibia can be efficiently derived from HR‐pQCT images and provide additional information regarding bone fragility. © 2010 American Society for Bone and Mineral Research