Immunotherapies targeting checkpoint molecule programmed cell death 1 (PD-1) protein were shown to be effective for treatment of non-Hodgkin lymphoma in people, but little is known about the expression of PD-1 or its ligand PD-L1 by canine lymphoma. Therefore, flow cytometry was used to analyse expression of PD-1 and PD-L1 in canine lymphoma, using fine-needle aspirates of lymph nodes from 34 dogs with B cell lymphoma (BCL), 6 dogs with T cell lymphoma (TCL) and 11 dogs that had relapsed. Furthermore, fine-needle aspirates were obtained from 17 healthy dogs for comparison. Lastly, the impact of chemotherapy resistance on expression of PD-1 and PD-L1 was assessed in vitro. These studies revealed increased expression of PD-L1 by malignant B cells compared to normal B cells. In the case of TCL, tumour cells and normal T cells both showed low to negative expression of PD-1 and PD-L1. In addition, tumour infiltrating lymphocytes from both BCL and TCL had increased expression of both PD-1 and PD-L1 expression compared to B and T cells from lymph nodes of healthy animals. In vitro, chemotherapy-resistant BCL and TCL cell lines exhibited increases in both PD-1 and PD-L1 expression, compared to non-chemotherapy selected tumour cells. These findings indicate that canine lymphomas exhibit upregulated checkpoint molecule expression, though the impact of checkpoint molecule expression on tumour biological behaviour remains unclear.