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Background Skin marker-based three-dimensional kinematic gait analysis were commonly used to assess the functional performance and movement biomechanics of the pelvic limb in dogs. Unfortunately, soft tissue artefact would compromise the accuracy of the reproduced pelvic limb kinematics. Multibody kinematics optimization framework was often employed to compensate the soft tissue artefact for a more accurate description of human joint kinematics, but its performance on the determination of canine pelvic limb skeletal kinematics has never been evaluated. This study aimed to evaluate a multibody kinematics optimization framework used for the determination of canine pelvic limb kinematics during gait by comparing its results to those obtained using computed tomography model-based fluoroscopy analysis. Results Eight clinically normal dogs were enrolled in the study. Fluoroscopy videos of the stifle joint and skin marker trajectories were acquired when the dogs walked on a treadmill. The pelvic limb kinematics were reconstructed through marker-based multibody kinematics optimization and single-body optimization. The reference kinematics data were derived via a model-based fluoroscopy analysis. The use of multibody kinematics optimization yielded a significantly more accurate estimation of flexion/extension of the hip and stifle joints than the use of single-body optimization. The accuracy of the joint model parameters and the weightings to individual markers both influenced the soft tissue artefact compensation capability. Conclusions Multibody kinematics optimization designated for soft tissue artefact compensation was established and evaluated for its performance on canine gait analysis, which provided a further step in more accurately describing sagittal plane kinematics of the hip and stifle joints.