ABSTRACT: Pythiosis in felines is a rare disease associated with the oomycete Pythium insidiosum. The aim of this report was to describe the macroscopic, histopathological, and molecular characteristics of P. insidiosum infection in a 2-year-old cat, with a localized invasive subcutaneous mass. The feline had an increase of volume near the anal region since it was younger. The cat died just after surgery. The necropsy was performed, and samples were collected for histopathological examination. Microscopically, the skin lesion was characterized by necro-eosinophilic dermatitis, panniculitis, and myositis surrounding negatively stained hyphal structures. In the sections stained with GMS, dark brown hyphae were clearly seen inside the affected tissue. They were rarely septate and their walls were almost parallel. Immunohistochemistry using a polyclonal anti-P. insidiosum antibody showed a strongly immunostained hyphae into the lesions. The analysis based on PCR had a positive result for P. insidiosum. Pythiosis should be considered in the differential diagnosis of subcutaneous tissue disorders in felines