Anterior cruciate ligament (ACL) reconstruction using a tendon graft remains the standard of care for ACL injuries. Postoperatively, the graft undergoes a biologic transition from tendinous to ligamentous in appearance. Despite substantial research efforts, little is known about the human ACL graft ligamentization process. Much of the current knowledge on graft ligamentization have been derived from biopsy studies. However, biopsies are invasive and suffer from sampling error. Our study demonstrates the feasibility and reliability of diffusion tensor imaging (DTI) for visualization and quantification of the ACL graft and supports its potential to serve as a biomarker to assess graft maturity.