Mast cell tumours (MCTs) are the most frequent cutaneous neoplasia of the dog, and they have very variable biological behaviour and survival times. Surgery is still the best treatment, and despite the several adjuvant therapies described, many cases are very aggressive and resistant to these treatments making it urgent to find new therapeutic targets. Nowadays, immunotherapy targeting immune checkpoints has been described as a complementary treatment for several human cancers, but it is still very scarcely studied in veterinary medicine. Therefore, this study aimed to investigate the expression of the checkpoint proteins programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) to evaluate their potential as therapeutic targets for MCT. Through immunohistochemical study, it was analysed the expression of PD-L1 and CTLA-4 in 74 MCT cases from the archive of the Veterinary Pathology Laboratory of the University of Porto (LabPatVet). Tumour size, histological grade, ki-67 proliferation index, mitotic count and presence of metastatic disease were also assessed. Most of the cases expressed both immune checkpoints in neoplastic cells. There was a statistically significant inverse association between the expression of CTLA-4 and MCT grade (p <
0,001) and mitotic count (p <
0.001). PD-L1 was significantly and negatively related to HG (p = 0.004), and tumour size (р = 0.014). Tumour size, histological grade and mitotic count were positively associated with metastatic disease. Additionally, it was observed that the expression of PD-L1 and CTLA-4 was interrelated (p <
0.001). This study demonstrated that MCT cells express both PD-L1 and CTLA-4 and that their expression was associated with MCT prognostic factors.