Purpose <p>Most commonly, the ACL is described as having two distinct anatomical bundles: anteromedial and posterolateral. These bundles have been observed in a gross anatomical setting, but not as closely observed histologically. Here, we sought to compare observations of ACL bundle number when observed anatomically versus histologically to determine if the double bundle ACL is a reliably observed structure.</p> Methods <p>Nineteen knees were dissected to expose the intracapsular ligaments. Bundle number was assessed in three ways: Via gross anatomical observation, histological observation, and via the presence of a connective tissue septum when observed histologically. Inter-rater agreements were calculated for each of the three modes. Cronbach’s alpha was calculated to assess the consistency of double-bundle observations across all three modes of observation.</p> Results <p>Prevalence of a double-bundle ACL under gross observation was 68.4%. Inter-rater agreement was fair (Fleiss’s kappa = 0.202). Under histological observation, prevalence of a double-bundle ACL was 47.4%, with only 26.4% demonstrating a connective tissue septum that could be said to divide the ligament.</p> Conclusion <p>These results suggest that the human ACL may exhibit more anatomical variation than has been previously appreciated in human cadaveric studies. Inter-rater agreement was fair, indicating that the demarcation of ACL bundles is not an obvious feature, even under gross observation. Additionally, our histological observations support the idea that ACL bundles exhibit substantial variation in terms of degree of distinct separation. This is, to our knowledge, the first study that has directly compared gross and microscopic observations of ACL bundle number.</p>