ABSTRACT.Our study describes the clinical, epidemiologic, pathologic, immunohistochemical, and molecular aspects (PCR) of a case of cutaneous pythiosis in a donkey (Equus asinus) from Brazil. During a dry period, the animal grazed for 4 months around a pond where the vegetation remained green. Skin lesions were nodular, multifocal, and disseminated, mainly involving the legs, ventral chest, and mammary gland. On cut surface, there were multifocal to coalescent discrete yellow foci, and occasional small cavitations with a few kunkers. Ulcerative nodular pyogranulomatous and eosinophilic dermatitis with folliculitis and furunculosis were observed histologically. Hyphae were observed in sections stained with Gomori methenamine silver. Immunohistochemistry with Pythium insidiosum antibodies yielded strong immunostaining of hyphae. P. insidiosum DNA was extracted from tissues in paraffin blocks by amplification of a fragment of 105 bp, which targets the 5.8S ribosomal gene. After the diagnosis of pythiosis, the larger skin lesions were excised and treated as second intention healing wounds, which were completely healed 30 days after resection. Small skin lesions regressed spontaneously in ~60 days. The granulomatous inflammation and outcome of the disease in this donkey were similar to cases of pythiosis in cattle.© 2016 The Author(s)