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Prototheca zopfii is an alga that can cause disease in animals and humans. Here, we reported a case of systemic protothecosis in a 2-year-old female Boxer. The animal exhibited tetraparesis and vertical nystagmus. It died two weeks after the onset of clinical signs and was necropsied. At gross examination, whitish areas were identified in the heart. Oval or round structures were observed by microscopic examination, some of which formed morula-like structures compatible with algae in the heart, skeletal muscle, and brain. Growth of Prototheca sp. was observed in the heart after culture at 37°C on Sabouraud agar medium with chloramphenicol. Immunohistochemical analysis of the heart and brain using an anti-Prototheca zopfii polyclonal antibody yielded positive results. Genotyping of the cultured agent from brain and heart samples was performed by restriction fragment length polymorphism of a specific 18S rDNA fragment. P. zopfii genotype 2 was reported to be the cause of disseminated protothecosis in this dog with manifestation in the brain, heart, and skeletal muscle.