OBJECTIVE To measure programmed cell death ligand-1 (PD-L1) mRNA expression in archived primary nodal diffuse large B-cell lymphoma (DLBCL) specimens of dogs and determine whether that expression was associated with progression-free survival time (PFST). SAMPLE Archived tumoral lymph node specimens from 42 dogs with DLBCL and lymph node specimens from 10 healthy dogs (controls). PROCEDURES Archived tumoral and control lymph node specimens underwent multiplex qPCR analysis with probes and primers against canine PD-L1 and glyceraldehyde 3-phosphate dehydrogenase (housekeeping gene) to determine PD-L1 mRNA expression. The 2-ΔΔCt method was used to calculate the fold change in PD-L1 expression in DLBCL specimens relative to that in control lymph nodes. Kaplan-Meier and Cox proportional hazard analyses were used to evaluate the association of various tumoral and clinical factors with PFST. RESULTS The fold change in PD-L1 mRNA expression in DLBCL specimens relative to control specimens ranged from 0.21 to 7.44. Twenty-one of 42 (50%) DLBCL specimens had a PD-L1-fold change > 1, which suggested PD-L1 was overexpressed in those specimens. Median PFST was 249 days for dogs with DLBCL. The PFST was not associated with PD-L1 mRNA expression but was associated with thrombocytopenia at the time of diagnosis (hazard ratio, 2.56; 95% confidence interval, 1.28 to 5.15). CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that tumoral PD-L1 mRNA expression varied among dogs with DLBCL and that PD-L1 MRNA was overexpressed in half the study population. Therefore, anti-PD-L1 therapies may be clinically beneficial for some dogs with DLBCL.