Immunohistochemistry features and cross-reactivity of commercially available antibodies are poorly understood in avian species. Here, we present a confirmative diagnosis of a rare systemically metastatic rhabdomyosarcoma in a seven-month-old male domestic chicken (Gallus domesticus). The animal had multiple grey to dark-red masses in the pectoral muscle, coelomic cavity, and multiple visceral organs. Microscopically, these masses were composed of spindloid neoplastic cells arranged into interwoven and patternless arrays with the presence of binucleated to multinucleated and karyomegalic neoplastic cells. A systemically metastatic rhabdomyosarcoma associated with avian leukosis viruses (ALV) infection was diagnosed by immunohistochemically demonstrating neoplastic cells dispersedly positive for desmin and p27 major capsid protein of ALVs, but diffusely negative for cytokeratin, vimentin, [Formula: see text]-smooth muscle actin, MyoD1, S-100, and neuron-specific enolase. These tumors and internal organs were further characterized positive for subgroups A, B, C, D, or E ALVs, but negative for subgroup J ALV, by polymerase chain reactions. The report suggests the potential of using IHC for a confirmative diagnosis of rhabdomyosarcoma and ALV infection in chickens. The role of endogenous and exogenous ALVs in the rhabdomyosarcoma is also discussed in the present report.