Technical Abstract: Several species of free-living amoebe can cause encephalomyelitis in animals and humans. Disseminated acanthamoebiasis was diagnosed in pyogranulomatous lesions in brain, thyroid, pancreas, heart, lymph nodes, and kidney of a one-year-old dog. Acanthamoeba sp. was identified in canine tissues by conventional histology, by immunofluorescence, by cultivation of the parasite from the brain of the dog that had been stored at -70'c for two months, and by PCR. The sequence obtained from the PCR product from the amoeba from the dog was compared to other sequences in the Acanthamoeba ribosomal DNA database and was determined to be genotype T1, associated with other isolates of Acanthamoeba obtained from granulomatous amebic encephalitis infections in humans.