Canine rhabdomyosarcoma (RMS) presents a diagnostic challenge due to its overlapping histologic features with other soft tissue sarcomas. The diagnosis of RMS currently relies on positive immunohistochemical (IHC) labeling for desmin; however, desmin expression is also observed in non-RMS tumors. Myogenin and MyoD1 are transcription factors reported to be sensitive and specific IHC markers for human RMS, but they are not widely used in veterinary oncology. The goals of this study were to develop an IHC protocol for myogenin and MyoD1, evaluate myogenin and MyoD1 labeling in canine RMS, and report clinical outcomes. Sixteen cases of possible RMS were retrospectively evaluated. A diagnosis of RMS was confirmed in 13 cases based on histological features and immunolabeling for myogenin and MyoD1, with the aid of electron microscopy in 2 cases. Desmin was negative in 3 cases of RMS. Two cases were of the sclerosing variant. The median age of dogs with RMS was 7.2 years. Anatomic tumor locations included previously reported sites such as bladder, larynx, heart, and orbit, as well as other locations typical of soft tissue sarcomas. Survival ranged from 47 to 1480 days for 5 dogs with available data. This study demonstrated that MyoD1 and myogenin should be included with desmin as part of a diagnostic IHC panel for canine RMS. Utilization of these antibodies to improve the accuracy of canine RMS diagnosis will ultimately allow for better characterization of the biological behavior and clinical outcomes of this disease, providing the groundwork for future comparative investigations in canine RMS.