Introduction: Triosephosphate isomerase 1 (TPI-1) is a glycolytic enzyme considered to be a marker of tumour growth. There are few reports of its expression in animal neoplasia, therefore the aim of the present study was to examine the expression of this protein in three types of canine cancer: squamous cell carcinoma, sweat gland adenocarcinoma and mammary carcinoma. Materials and Methods: Archival paraffin wax-embedded tissues of canine squamous cell carcinoma (n = 10), sweat gland adenocarcinoma (n = 8), mammary carcinoma (n = 5), as well as non-neoplastic skin (n = 4) and mammary gland (n = 3) were analysed by immunohistochemistry using commercial goat polyclonal anti-TPI-1 antibody (LifeSpan Biosciences) and the DAB /HRP system (Dako) for ‘visualization’ of labelling. Results: In all of the squamous cell carcinomas, the sweat gland adenocarcinomas and mammary gland carcinomas, moderate to strong positive nuclear reactivity was observed in neoplastic cells, single lymphoid cells and in activated fibroblasts of connective tissue. The strongest reaction was visible in the least-differentiated neoplastic cells. In non-neoplastic tissues, strong positive nuclear labelling was present in the external root sheaths of hair follicles, the basal layer of the epidermis and in single lymphoid cells within the dermis, while the sweat glands and the mammary glands showed either no positive reaction or only a faint labelling of cell nuclei. Discussion: The results of the experiment appear to confirm an association of TPI-1 protein with neoplastic growth and proliferation, which was previously reported in various human cancers, in all three types of canine malignant tumours.