The purpose of this study was to estimate and compare the moment arm length of the patellar tendon (d) during passive knee extension using three different reference landmarks; instant centre of rotation (ICR), tibiofemoral contact point (TFCP) and geometrical centre of the posterior femoral condyles (GCFC). Measurements were taken on the right leg on seven healthy males during passive knee rotation performed by the motor of a Cybex Norm isokinetic dynamometer. Moment arms lengths were obtained by analysing lateral X-ray images recorded using a GE FlexiView 8800 C-arm videofluoroscopy system. The d-knee joint angle relations with respect to GCFC and ICR were similar, with decreasing values from full knee extension (~5.8 cm for d (GCFC) and ~5.9 cm for d (ICR)) to 90 degrees of knee flexion (~4.8 cm for both d (GCFC) and d (ICR)). However, the d (TFCP)-knee joint angle relation had an ascending-descending shape, with the highest d (TFCP) value (~5 cm) at 60 degrees of knee flexion. There was no significant difference between the GCFC and ICR methods at any knee joint angle. In contrast, there were significant differences (P < 0.01) between d (ICR) and d (TFCP) at 0 degrees , 15 degrees , 30 degrees and 45 degrees of knee flexion and between d (GCFC) and d (TFCP) at 0 degrees , 15 degrees and 30 degrees of knee flexion (P < 0.01). This study shows that when using different knee joint rotation centre definitions, there are significant differences in the estimates of the patellar tendon moment arm length, especially in more extended knee joint positions. These differences can have serious implications for joint modelling and loading applications.