BackgroundThe Gait Deviation Index (GDI) is a metric clinicians use to assess overall gait pathology in children with cerebral palsy (CP) by comparing kinematic data to a normative sample. The Gait Variability Index (GVI) is a related metric that quantifies the variability in spatio-temporal variables during gait. The GDI and GVI have been verified using marker-based motion capture approaches, but video-based markerless motion capture has not been compared using these tools in children with CP. Research questionDo GDI and GVI scores differ when measured using markerlessTheia3D and a marker-based approach between the more and less affected legs in children with CP? MethodsFifteen children with CP (GMFCS levels I-IV) and 24 typically developing children aged 6–18 years were recruited for this study. Overground walking was performed at a self-selected pace while the pelvis and lower limb kinematics were simultaneously recorded using both motion capture systems. Differences in GDI and GVI scores when considering the effect of system and limb impairment were analyzed using two-way repeated-measures ANOVAs. ResultsGDI scores were 6.9 points lower (p < 0.05) when measured using Theia3D compared to the marker-based approach and 6.8 points lower (p < 0.05) in the more affected limbs than in the less affected limbs. These GDI score differences are considered clinically significant. No differences were identified in GVI scores between systems or limb impairment. Differences in kinematic measurements were found in children with CP, including pelvic tilt, hip flexion/extension, hip rotation, and foot progression angle, where root mean square differences between systems exceeded 10°. SignificanceTheia3D can adequately measure variability in spatio-temporal gait parameters for quantifying GVI scores in children with CP compared to the marker-based approach. However, caution is needed when quantifying lower limb kinematics and interpreting GDI and GVI scores using Theia3D in children with CP.