Accurate tunnel placement is important for a successful anterior cruciate ligament (ACL) reconstruction. Controversy exists concerning the preferred method of femoral tunnel preparation, with proponents of both medial portal and transtibial drilling techniques. Current ACL literature suggests that placement of the femoral ACL attachment site posterior or “low” in the ACL footprint leads to more anatomically correct ACL mechanics and better rotational control. There is limited literature focusing on ACL graft displacement through knee range of motion based on specific paired placement of femoral and tibial tunnels. Our purpose was to assess ACL isometry between multiple combinations of femoral and tibial tunnels. We hypothesized that placement of the graft at the posterior aspect of the ACL footprint on the femur would be significantly less isometric and lead to more graft displacement as compared with central or anterior placement. The ACL of matched pairs of cadaveric knees was arthroscopically debrided while leaving the soft tissue footprint on the femur and tibia intact. One knee from each pair underwent notchplasty. In all knees, three femoral and three tibial tunnels were created at the anterior, central, and posterior aspects of the ACL footprint. A suture was passed through each tunnel combination (nine potential pairs), and the change in isometry was measured throughout full knee range of motion. Placement of the femoral tunnel along the posterior aspect of the ACL footprint was less isometric compared with a central or anterior position in the femoral footprint. Placement of a posterior tibial tunnel also led to decreased isometry, but tibial tunnel placement affected isometry to a lesser extent than femoral tunnel placement. The combination of a posterior femoral and posterior tibial tunnel resulted in greater than 1 cm of graft excursion from full flexion to extension. Placement of ACL tunnels at anisometric sites may adversely affect the mechanical properties and behavior of the ACL graft, resulting in either graft laxity in flexion or overconstraint and loss of extension.