Objective-To characterize biomechanical differences in gait between dogs with and without an amputated thoracic limb. Animals-Client-owned dogs (16 thoracic-limb amputee and 24 quadruped [control] dogs). Procedures-Dogs were trotted across 3 in-series force platforms. Spatial kinematic and kinetic data were recorded for each limb during the stance phase. Results-Amputees had significant increases in stance duration and vertical impulse in all limbs, compared with values for control dogs. Weight distribution was significantly increased by 14% on the remaining thoracic limb and by a combined 17% on pelvic limbs in amputees. Braking ground reaction force (GRF) was significantly increased in the remaining thoracic limb and pelvic limb ipsilateral to the amputated limb. The ipsilateral pelvic limb had a significantly increased propulsive GRF. The carpus and ipsilateral hip and stifle joints had significantly greater flexion during the stance phase. The cervicothoracic vertebral region had a significantly increased overall range of motion (ROM) in both the sagittal and horizontal planes. The thoracolumbar vertebral region ROM increased significantly in the sagittal plane but decreased in the horizontal plane. The lumbosacral vertebral region had significantly greater flexion without a change in ROM. Conclusions and Clinical Relevance-Compared with results for quadruped dogs, the vertebral column, carpus, and ipsilateral hip and stifle joints had significant biomechanical changes after amputation of a thoracic limb. The ipsilateral pelvic limb assumed dual thoracic and pelvic limb roles because the gait of a thoracic limb amputee during trotting appeared to be a mixture of various gait patterns.