Fluorine-18-fluoro-2-deoxy-d-glucose positron emission tomography (FDG PET) provides physiologic images of tissues based on their glucose metabolism. The combination of FDG PET and CT (FDG PET-CT) has been utilized in human musculoskeletal imaging to localize soft tissue lesions, however, this modality has not been thoroughly investigated for the diagnosis of canine lameness. This prospective, descriptive study evaluated FDG PET-CT findings in 25 client-owned dogs with inconclusive origin of thoracic or pelvic limb lameness (thoracic limb n = 15/25, 60%; pelvic limb n = 6/25, 24%; and combination of both limbs n = 4/25, 16%). We hypothesized that FDG PET-CT would aid the detection of soft tissue lesions not visible with other imaging modalities. Combined FDG PET-CT detected soft tissue lesions in 40% (n = 10/25) and osteoarthritis in 64% (n = 16/25) of the patients. FDG PET detected more soft tissue lesions than contrast-enhanced CT (n = 15/15, 100% and n = 12/15, 80%, respectively), while CT identified more osteoarthritis lesions than FDG PET (n = 26/26, 100% and n = 18/26, 69%, respectively). The three imaging-diagnoses based on the FDG PET component included the following: flexor carpi ulnaris muscle tear, psoas major myopathy, and tarsal desmopathy. No diagnosis for the lameness was obtained in three dogs. Findings supported FDG PET-CT as a useful adjunct imaging modality for detection of certain soft tissue injuries of the musculoskeletal system. Combined FDG PET-CT should be considered for cases where the cause of lameness is thought to be of soft tissue origin and cannot be diagnosed by conventional means.