The study design is a prospective, case-control. The aim of this study was to develop a reliable measurement technique for the assessment of lumbar spine kinematics using digital video fluoroscopy in a group of patients with low back pain (LBP) and a control group. Lumbar segmental instability (LSI) is one subgroup of nonspecific LBP the diagnosis of which has not been clarified. The diagnosis of LSI has traditionally relied on the use of lateral functional (flexion-extension) radiographs but use of this method has proven unsatisfactory.Fifteen patients with chronic low back pain suspected to have LSI and 15 matched healthy subjects were recruited. Pulsed digital videofluoroscopy was used to investigate kinematics of lumbar motion segments during flexion and extension movements in vivo. Intersegmental linear translation and angular displacement, and pathway of instantaneous center of rotation (PICR) were calculated for each lumbar motion segment. Movement pattern of lumbar spine between two groups and during the full sagittal plane range of motion were analyzed using ANOVA with repeated measures design. Intersegmental linear translation was significantly higher in patients during both flexion and extension movements at L5-S1 segment (p < 0.05). Arc length of PICR was significantly higher in patients for L1-L2 and L5-S1 motion segments during extension movement (p < 0.05). This study determined some kinematic differences between two groups during the full range of lumbar spine. Devices, such as digital videofluoroscopy can assist in identifying better criteria for diagnosis of LSI in otherwise nonspecific low back pain patients in hope of providing more specific treatment.