Reconstruction of the anterior cruciate ligament (ACL) allows to restore stability of the knee, in order to facilitate the return to activity (RTA). Although it is understood that the tendon autograft undergoes a ligamentous transformation postoperatively, knowledge about longitudinal microstructural differences in tissue integrity between types of tendon autografts (ie, hamstring vs. patella) remains limited. Diffusion tensor imaging (DTI) has emerged as an objective biomarker to characterize the ligamentization process of the tendon autograft following surgical reconstruction. One major limitation to its use is the need for a pre-injury baseline MRI to compare recovery of the graft, and inform RTA. Here, we explore the relationship for DTI biomarkers (fractional anisotropy, FA) between knees bilaterally, in healthy participants, with the hypothesis that agreement within a patient's knees may support the use of the contralateral knee as a reference to monitor recovery of the tendon autograft, and inform RTA. Fifteen participants with no previous history of knee injuries were enrolled in this study (age, 26.7 +/− 4.4 years; M/F, 7/8). All images were acquired on a 3T Prisma Siemens scanner using a secured flexible 18-channel coil wrapped around the knee. Both knees were scanned. A 3D anatomical Double Echo Steady State (DESS) sequence was acquired on which regions of interest (ROI) were placed consistent with the footprints of the ACL (femur, posteromedial corner on medial aspect of lateral condyle; tibia, anteromedial to intercondylar eminence). Diffusion images were acquired using fat saturation based on optimized parameters in-house. All diffusion images were pre-processed using the FMRIB FSL toolbox. The footprint ROIs of the ACL were then used to reconstruct the ligament in each patient with fiber-based probabilistic tractography (FBPT), providing a semi-automated approach for segmentation. Average FA was computed for each subject, in both knees, and then correlated against one another using a Pearson correlation to assess the degree of similarity between the ACLs. A total of 30 datasets were collected for this study (1/knee/participant; N=15). The group averaged FA (+/− standard deviation) for the FBPT segmented ACLs were found to equal 0.1683 +/− 0.0235 (dominant leg) and 0.1666 +/− 0.0225 (non-dominant leg). When comparing both knees within subjects, reliable agreement was found for the FBPT-derived ACL with a linear correlation coefficient (rho) equal to 0.87 (P < 0 .001). We sought to assess the degree of concordance in FA between the knees of healthy participants with hopes to provide a method for using the contralateral “healthy” knee in the comparison of autograft-dependent longitudinal changes in microstructural integrity, following ACL reconstruction. Our results suggest that good agreement in anisotropy can be achieved between the non-dominant and dominant knees using DTI and the FBPT segmentation method. Contralateral anisotropy of the ACL, assuming no previous injuries, may be used as a quantitative reference biomarker for monitoring the recovery of the tendon autograft following surgical reconstruction, and gather further insight as to potential differences between chosen autografts. Clinically, this may also serve as an index to supplement decision-making with respect to RTA, and reduce rates of re-injuries