B cell high grade lymphoma is the most common hematopoietic malignancy in dogs. Although the immune checkpoint molecules, programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), and immune checkpoint inhibitors have been evaluated for the treatment of various human lymphoid malignancies, the expression of those molecules and their relationship with prognosis remain unknown in canine lymphoma. The objective of this study was to evaluate the expression of costimulatory molecules on peripheral blood lymphocytes and tumor infiltrating lymphocytes, in addition to associated ligand expression in the lymph nodes of patients with B cell multicentric high grade lymphoma. Eighteen patients diagnosed with B cell high grade lymphoma and nine healthy control dogs were enrolled. Flow cytometric analysis revealed that the expression of PD-1 on CD4+ peripheral and tumor infiltrating lymphocytes and CTLA-4 on CD4+ peripheral lymphocytes was significantly higher in the lymphoma group than in the control group. The expression level of CD80 mRNA was significantly lower in the lymphoma group than in the control group. In contrast, there were no significant differences in PD-L1, PD-L2, and CD86 expression between the groups. Dogs with CTLA-4 levels below the cutoff values, which were determined based on receiver operating characteristic curves, on peripheral CD4+, CD8+, and tumor infiltrating CD4+ lymphocytes had significantly longer survival than dogs with values above the cutoff. Although it is uncertain whether the expression of immune checkpoint molecules affect the biological behavior of canine lymphoma, one possible explanation is that PD-1 and CTLA-4 might be associated with the suppression of antitumor immunity in dogs with B cell high grade lymphoma, particularly through CD4+ T cells.