Background Prosthetic trochlear design is important to ideal postoperative patellofemoral kinematics and knee function. But there has been little research on the differences of trochlear groove trackings between the native and prosthetic knees. We aimed to investigate the differences between native and prosthetic knees through the entire trochlear length by three-dimensional computerized quantification.Methods Virtual total knee arthroplasty was performed, using three-dimensional models of 42 healthy knees that were matched to the femoral components of five different prosthesis systems. Coaxial planes were created along the trochlear groove in 3° increments, and the deepest points of the trochlear groove were marked in each plane. Taking the lower extremity mechanical axis as reference line, the differences in the mediolateral location of the groove tracking were analyzed between the native and prosthetic knees.Results From the proximal to the distal end, the native tracking started from 0° cross section and extended laterally and then medially with its turning point located in 69° cross section, while the prosthetic knees showed medial orientation throughout the trochlear length. Compared with the proximal portion of the native tracking, the prosthetic trackings extended along a paradoxical orientation and started from a more proximal and lateral position, with maximal discrepancy to, 3.2 mm in the 0° cross section. Distally, the prosthetic trackings were located significantly medial, with maximal discrepancy, to 2.4 mm in the 69° cross section.Conclusion The prosthetic trochlear design varies among different types, and does not conform to that of the native knee in terms of shape, orientation, and location, which may cause soft tissue tension imbalance and abnormal patellofemoral biomechanics during knee flexion. This study may be helpful for prosthetic trochlear design that accords with native anatomy so as to optimize patellofemoral biomechanics and decrease the risk of patellofemoral complications.