Introduction: The main adaptive immune cells are T and B lymphocytes and they play key roles in the induction of immune responses against canine mammary tumours. Investigating these cell subpopulations may lead to more precise diagnosis of these malignancies. Material and Methods: The percentages of CD3+ , CD4+ and CD8+ T cells and of CD21+ B cells in the peripheral blood of bitches with malignant mammary tumours were compared with those in the blood of healthy animals. The phenotypic features of peripheral blood leukocytes were evaluated by flow cytometry. Results: There was a significant difference in the mean percentages of CD3+ lymphocytes between healthy (66.7%) and metastatic dogs (46.1%), and between tumour-bearing nonmetastatic (66.6%) and metastatic dogs. There was also a significant difference in CD4+ T helper cell percentages between healthy dogs (40.4%) and dogs with metastases (23.2%), and between the latter and dogs without them (35.5%). In the case of CD21+ lymphocyte subsets, a significant difference was noted between healthy animals (10.9%) and those with metastases (20.1%), and between the latter and patients without metastases (8.5%). There were also significant differences in CD3+ /CD21+ ratios between the group with metastases (3.0), the healthy group (7.8), and the group without metastases (8.5). Similarly, a significant difference was noted in CD4+ /CD8+ ratios between animals with metastases (1.4), bitches in the control group (2.2), and dogs without metastases (1.9). Conclusion: Peripheral blood leukocyte phenotypic characteristics are putative novel biomarkers. These findings may be useful in future studies improving mammary tumour diagnostic procedures, especially in metastasis detection.