Introduction. Canine lymphoma remains one of the most chemotherapy-responsive neoplasia in dogs. Many factors affect the prognosis in dogs treated for lymphoma, but indications for a specific treatment regimen in individual animals with lymphoma are poorly defined. Topoisomerase IIα (TOPIIα) is a key enzyme in DNA replication and a molecular target for TOPIIα inhibitors, including anthracyclines. The aim of this study was to determine the expression of TOPIIα in canine malignant lymphomas. The relationship between TOPIIα expression in canine lymphomas and potential sensitivity of neoplastic cells to anthracycline-based chemotherapy is discussed. Materials and method. Samples of formalin-fixed paraffin-embedded lymph nodes from 47 dogs with different subtypes of non-Hodgkin’s (34 B-cell and 13 T-cell) lymphoma were immunohistochemically labeled with anti-TOPIIα. The number of positive cells and the intensity of the reaction were taken into account in order to assess TOPIIα expression. Results. TOPIIα expression was evident in all cases, although differences in the number of positive cells and intensity of the reaction were demonstrated between B-cell and T-cell lymphoma groups as well as within individual groups. Based on the established scoring system, in the B-cell lymphoma group statistically higher expression of TOPIIα was found compared to the T-cell lymphoma group (P = 0.006). In B-cell lymphoma group moderate (41.18%) and strong (32.35%) TOPIIα expression predominated, whereas among T-cell lymphoma group the majority were cases with a weak (46.15%) TOPIIα expression. Conclusion. These preliminary results indicate that further studies are needed to determine the prognostic value of TOPIIα expression with regard to the sensitivity of canine B-cell lymphomas to anthracycline-based chemotherapy regimen. Nevertheless, this study indicates the possibility of choosing the appropriate treatment of canine lymphoma based on TOPIIa expression.