In humans, passive immunotherapy with anti-CD20 monoclonal antibodies (mAbs) has created immeasurable improvements in outcomes of patients with B-cell malignancies. However, the lack of comparable reagents has precluded development of this approach in dogs. We developed a novel anti-canine CD20 mAb designated as 6C8. 6C8 recognized the extracellular domain of canine CD20 and showed high-affinity binding to canine CD20 in solution, as well as in its native conformation on canine B-cells. The 6C8 target was expressed invariably in B-cell lineage cells, but not in T-cells or myeloid cells. 6C8 promoted phagocytosis of B-cell lymphoma cells by macrophages, but in its current framework, it did not induce direct cytotoxicity or complement dependent cytotoxicity. In summary, we have established a novel anti-canine CD20 mAb that is useful as a diagnostic tool to phenotype B-cells, and which could be integrated as a tool for passive immunotherapy to treat dogs with B-cell disorders.