This study aimed to determine the test-retest reliability and inter-tester reliability of kinematic measures in a three-dimensional gait analysis system. Using a VICON 140™ three-dimensional motion analysis system, kinematic data for lower extremities during walking were collected by 2 testers (senior physical therapists) for 6 unimpaired adults (age = 20 to 52; mean = 35.2 ± 6.2). The study was conducted using a repeated measures design consisting of two testing sessions per day on two separate testing days. The reliability of joint angle data collected by two different testers on two different days was compared for 2 sessions (days) × 2 testers × 5 trials. Skin markers were placed on 15 defined pelvis and lower body locations in accordance with the VICON Clinical Manager model. Prior to the study commencing, the two physical therapists practiced marker placement for a 3 month period. The first measurements (T1) were carried out by two testers on the same day. The second measurement session (T2) was performed within two weeks using an identical procedure. Coefficients of multiple correlation (CMC) were calculated to evaluate the consistency between the kinematic variables across testers and sessions. Both test-retest and inter-tester reliability were high for motion in the sagittal plane (R a= 0.971 to 0.994), the frontal plane (R a= 0.759 to 0.977), and the transverse plane (R a= 0.729 to 0.899), excluding pelvic tilt. Reduction of variability of marker placement appears possible with standardization and understanding of the placement method. These findings provide evidence of the reliability of using three-dimensional motion analysis for measuring human gait.