Background: Antibody therapy targeting programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) is a promising therapy in human cancer, but only limited information on PD-L1 expression in canine tumors is available. Materials and Methods: PD-L1 expression was examined in 31 canine tumor cell lines of various origins by flow cytometry and western blotting, and in canine tumor and normal tissue specimens by immunohistochemistry. Results: PD-L1 was only expressed on the cell surface of a small number of cell lines but was found expressed within the cells of almost all cell lines. Immunohistochemistry revealed that PD-L1 is frequently expressed in malignant melanoma, mammary gland tumor, mast cell tumor and lymphoma, but less frequently in softtissue sarcoma and hemangiosarcoma. PD-L1 was also expressed in some of the cells of normal canine tissue specimens. Conclusion: Canine tumors with PD-L1 expression that were identified in this study are potential candidates for antiPD-1 and antiPD-L1 therapy.