The use of magnetic resonance imaging has been proposed by many investigators for establishment of joint reference systems and kinematic tracking of musculoskeletal joints. In this study, the intraobserver and interobserver reliability of a strategy to establish anatomic reference systems using manually selected fiducial points were quantified for seven sets of MR images of the human knee joint. The standard error of the measurement of the intraobserver and interobserver errors were less than 2.6°, and 1.2 mm for relative tibiofemoral orientation and displacement, respectively. An automated motion tracking algorithm was also validated with a controlled motion experiment in a cadaveric knee joint. The controlled displacements and rotations prescribed in our motion tracking validation were highly correlated to those predicted (Pearson’s correlation=0.99, RMS errors=0.39 mm, 0.38°). Finally, the system for anatomic reference system definition and motion tracking was demonstrated with a set of MR images of in vivo passive flexion in the human knee.