The choice of a reference posture is important when investigating rearfoot motion in clinical populations. The reference posture used may affect the magnitude of the peak angles and therefore may not enable comparison of the rearfoot kinematics across different populations. This study examined the relationship between the rearfoot frontal plane pattern of motion and three reference postures during the stance phase of walking in healthy and patellofemoral pain syndrome (PFPS) subjects. The three reference postures investigated were: Relaxed Standing posture, subtalar joint neutral position (STJN) and when the calcaneus and the lower leg were vertically aligned (Vertical Alignment). The rearfoot inversion/eversion during the stance phase was measured in 14 healthy subjects and 13 subjects with diagnosed PFPS using three dimensional motion analysis with the three different reference postures. The graphs of rearfoot inversion/eversion motion were overlaid with the angle at the rearfoot in the static posture and any intersection between the static angle and rearfoot motion was noted. An ANOVA showed significant differences in static posture between the groups for Relaxed Standing (p = 0.01), and STJN (p = 0.02). For both groups, with Relaxed Standing as a reference posture, the mean rearfoot pattern of motion did not intersect the Relaxed Standing static angle during the stance phase. The use of Vertical Alignment reference posture, however, showed an intersection of this reference posture through the rearfoot pattern of motion. The use of the Vertical Alignment reference posture also generated a typical rearfoot motion pattern for both groups and therefore it may be an appropriate reference posture for both healthy and PFPS individuals.