Background: Medial patellofemoral ligament (MPFL) reconstruction is associated with a high rate of complications, including recurrent instability and persistent knee pain. Technical errors are among the primary causes of these complications. Understanding the effect of adjusting patellofemoral attachments on length change patterns may help surgeons to optimize graft placement during MPFL reconstruction and to reduce graft failure rates. Purpose: To determine the in vivo length changes of the MPFL during dynamic, weightbearing motion and to map the isometry of the 3-dimensional wrapping paths from various attachments on the medial femoral epicondyle to the patella. Study Design: Descriptive laboratory study. Methods: Fifteen healthy participants were studied with a combined computed tomography and biplane fluoroscopic imaging technique during a lunge motion (full extension to ~110° of flexion). On the medial femoral epicondyle, 185 attachments were projected, including the anatomic MPFL footprint, which was divided into 5 attachments (central, proximal, distal, posterior, and anterior). The patellar MPFL area was divided into 3 possible attachments (proximal, central, and distal). The length changes of the shortest 3-dimensional wrapping paths of the various patellofemoral combinations were subsequently measured and mapped. Results: For the 3 patellar attachments, the most isometric attachment, with an approximate 4% length change, was located posterior and proximal to the anatomic femoral MPFL attachment, close to the adductor tubercle. Attachments proximal and anterior to the isometric area resulted in increasing lengths with increasing knee flexion, whereas distal and posterior attachments caused decreasing lengths with increasing knee flexion. The anatomic MPFL was tightest in extension, decreased in length until approximately 30° of flexion, and then stayed near isometric for the remainder of the motion. Changing both the femoral and patellar attachments significantly affected the length changes of the anatomic MPFL ( P < .001 for both). Conclusion: The most isometric location for MPFL reconstruction was posterior and proximal to the anatomic femoral MPFL attachment. The anatomic MPFL is a dynamic, anisometric structure that was tight in extension and early flexion and near isometric beyond 30° of flexion. Clinical Relevance: Proximal and anterior MPFL tunnel positioning should be avoided, and the importance of anatomic MPFL reconstruction is underscored with the results found in this study.