The femoral footprint of the anterior cruciate ligament (ACL) is a much-studied anatomic structure, predominantly due to its importance during ACL reconstruction surgery. A new technique utilising high-resolution micro-computed tomography (micro-CT) is described, allowing detailed three-dimensional (3D) quantitative analysis of this structure. Seven cadaveric knees were scanned using micro-CT, yielding 3D data with a reconstructed voxel size of 60 μm. A novel method of 3D surface extraction was developed and validated, facilitating both qualitative observation of surface details and quantitative topographic assessment using colour-coded relief maps. Images were displayed on an immersive 3D visualisation wall, and ten experienced ACL clinicians were surveyed as to the presence and morphology of osseous landmarks, providing qualitative assessment of whether such features can be reliably identified for navigation during surgery. Both quantitative analysis and qualitative assessment of the footprints in this study showed significant variability in the presence and morphology of osseous landmarks, with the lateral intercondylar ridge being objectively present in four out of seven relief maps, although reportedly seen in six out of seven cases in the qualitative study, suggesting an element of subjectivity and interpretation. This is the first study to utilise micro-CT in the study of ACL anatomy.