Objective: High-resolution peripheral quantitative computed tomography (HR-pQCT)is a novel imaging modality capable of visualizing bone microarchitecture in vivo at human peripheral sites such as the distal radius and distal tibia. This research has extended the technology to provide a non-invasive assessment of bone microarchitecture at the human knee by establishing new hardware, imaging protocols and data analysis. Design: A custom leg holder was developed to stabilize a human knee centrally within a second generation HRpQCT field of view. Five participants with anterior cruciate ligament reconstructions had their knee joint imaged in a continuous scan of 6 cm axially. The nominal isotropic voxel size was 60.7 mu m. Bone mineral density and microarchitecture were assessed within the weight-bearing regions of medial and lateral compartments of the knee at three depths from the weight-bearing articular bone surface, including both the cortical and trabecular bone regions. Results: Scan duration was approximately 18 min per knee and produced 5 GB of projection data and 10 GB of reconstructed image data (2304 x 2304 image matrix, 1008 slices). Motion during the scan was minimized by the leg holder and was similar in magnitude as a scan of the distal tibia. Bone mineral density and microarchitectural parameters were assessed for 16 volumes of interest in the tibiofemoral joint. Conclusions: This is a new non-invasive in vivo assessment tool for bone microarchitecture in the human knee that provides an opportunity to gain insight into normal, injured and surgically reconstructed human knee bone architecture in cross-sectional or longitudinal studies. Crown Copyright (C) 2017 Published by Elsevier Inc. All rights reserved.