High-resolution peripheral quantitative computed tomography (HR-pQCT) provides in vivo three-dimensional (3D) imaging at the distal radius and tibia and has been increasingly used to characterize cortical and trabecular bone morphology in clinical studies. In this study, we comprehensively examined the accuracy of HR-pQCT and HR-pQCT based micro finite element (μFE) analysis predicted bone elastic stiffness and strength through comparisons with gold-standard micro computed tomography (μCT) based morphological/μFE measures and direct mechanical testing results. Twenty-six sets of human cadaveric distal radius and tibia segments were imaged by HR-pQCT and μCT. Microstructural analyses were performed for the registered HR-pQCT and μCT images. Bone stiffness and yield strength were determined by both HR-pQCT and μCT based linear and nonlinear μFE predictions and mechanical testing. Our results suggested that strong and significant correlations existed between the HR-pQCT standard, model-independent and corresponding μCT measurements. HR-pQCT based nonlinear μFE overestimated stiffness and yield strength while the linear μFE underestimated yield strength, but both were strongly correlated with those predicted by μCT μFE and measured by mechanical testing at both radius and tibia (R2>0.9). The microstructural differences between HR-pQCT and μCT were also examined by the Bland–Altman plots. Our results showed HR-pQCT morphological measurements of BV/TVd, Tb.Th, and Tb.Sp, can be adjusted by correction values to approach true values measured by gold-standard μCT. In addition, we observed moderate correlations of HR-pQCT biomechanical and microstructural parameters between the distal radius and tibia. We concluded that morphological and mechanical properties of human radius and tibia bone can be assessed by HR-pQCT based measures.