Earlier, we described a breed-specific inflammatory myopathy in Dutch Kooiker dogs (Het Nederlandse Kooikerhondje), one of the nine Dutch breeds. The disease commonly manifests itself with clinical signs of difficulty walking, muscle weakness, exercise intolerance, and/or dysphagia. In nearly all dogs’ creatine kinase (CK) activity was elevated. Histopathology reveals the infiltration of inflammatory cells within the skeletal muscles. The objective of this study was to further investigate and characterize the histopathological changes in muscle tissue and immunophenotype the inflammatory infiltrates. FFPE fixed-muscle biopsies from 39 purebred Kooiker dogs were included and evaluated histopathologically according to a tailored classification scheme for skeletal muscle inflammation. As in other breed-related inflammatory myopathies, multifocal, mixed, and predominantly mononuclear cell infiltration was present, with an initial invasion of viable muscle fibres and the surrounding stroma leading to inflammation, necrosis, and tissue damage. Immunophenotyping primarily revealed lymphohistiocytic infiltrates, with CD3+ T-cells being the predominant inflammatory cell type, accompanied by CD8+ cytotoxic T-cells. The concurrent expression of MHC-II class molecules on myofibres suggests their involvement in initiating and maintaining inflammation. Additionally, CD20+ B-cells were identified, though in lower numbers compared to T-cells, and IBA-1-positive macrophages were frequently seen. These findings suggest a breed-specific subtype of polymyositis in Kooiker dogs, akin to other breeds. This study sheds light on the immune response activation, combining adaptive and innate mechanisms, contributing to our understanding of polymyositis in this breed.