We report a case of high-grade undifferentiated pleomorphic sarcoma in a free-living white-eared opossum (Didelphis albiventris) in Central Brazil. Grossly, it appeared as a soft yellow mass, approximately 8 cm in diameter, with an irregular, adherent and ulcerated surface, which extended from the proximal epiphysis of the femur to the proximal third of the tibia and fibula. The mass invaded the medullary cavity and had a necrotic centre. Multiple nodular metastases (0.5–1.5 cm) were present in the liver, lungs and spleen. Histologically, the neoplasm was characterized by pleomorphic cells with indistinct boundaries, many multinucleated cells, necrosis and tissue invasion. There was intense cytoplasmic immunolabelling of neoplastic cells for vimentin and moderate cytoplasmic and nuclear labelling for human muscle actin, human smooth muscle actin and lysozyme. Immunolabelling for Iba1, CD18, CD1A, cytokeratin AE1/AE3, glial fibrillary acidic protein and desmin was negative. To our knowledge, this is the first report of an undifferentiated pleomorphic sarcoma in this species.