Canine mammary tumours (CMTs) are naturally occurring cancer that it is frequent in intact female dogs, and rare in male dogs. The annual reported incidence is approximately 198 cases of every 100,000 female dogs, with this incidence tending to be higher in regions of the world where early spaying is seldom practised. The exact cause of CMTs is still unknown, even though myriads of risk factors have been attributed. The milestone for prognosis in CMTs is an early diagnosis, with late diagnosis being usually associated with low survival rates after the surgical treatment. Unfortunately, CMTs may be associated with non-specific clinical signs, which makes early detection by the owner, or during routine physical examinations difficult. Like in human oncology, recent advances made in the diagnosis of CMTs include the discovery of biomarkers in both mammary gland tissues by immunohistochemistry techniques and blood/plasma/serum by other molecular methods. So far in veterinary oncology, only a few biomarkers have been discovered and standardized for use. Therefore, we hypothesized that biomarkers in the serum and mammary tissue can be used in the diagnosis of CMTs. The objectives of the present study were two-fold. Firstly, the serum of female dogs was assessed in the determination of suitable biomarkers for CMTs. To this end, blood was sampled from 30 female dogs and subsequently centrifuged. The collected serum was analysed through CD antibody microarrays targeting 90 CD surface markers and 56 cytokines/chemokines. A total of five CD proteins, namely CD20, CD45RA, CD53, CD59, and CD99, were selected due to their differential protein abundance in relation to either log FC values or p-values. These were further analysed utilizing immunoblotting techniques to validate the microarray results. CD45RA showed a significantly lower (P<0. 05) abundance in the serum obtained from the individuals presenting mammary neoplasia in comparison to the control group. In contrast, CD99 resulted significantly more abundant in neoplastic female dogs than in healthy patients. Finally, CD20 showed a significantly higher abundance in individuals carrying a malignant mammary tumour in comparison to healthy patients, although no differential expression between malignant and benign tumours was observed. It was concluded that both CD99 and CD45RA are indicators of mammary tumour presence, although without differentiating between malignant and benign canine mammary neoplasia. Secondly, the present study aimed to assess the gene expression mRNA in mammary tissue of both healthy and diagnosed CMT female dogs through quantitative PCR (RT-qPCR). The target genes chosen for this study included vascular endothelial growth factor A (VEGFa), CD20, progesterone receptor (PGR), hyaluronidase 1 (HYAL1), programmed death ligand 1 (PDL1), epidermal growth factor (EGF), relaxin 2 (RLN2), and matrix metallopeptidase 3 (MMP3). Fifty-eight mammary tissue samples from female dogs, both healthy (n=3) and presenting mammary tumours (n-58), were analysed for this purpose. Transcription levels of HYAL1, VEGFa, CD20 and PGR genes showed similar higher expressions in both benign and malignant mammary tumours in comparison to healthy individuals, whereas MMP3, EGF, PDL-1, and RLN2 genes were found at higher expression levels in malignant tumours than in the benign tumours and the control group. Therefore, it was concluded that HYAL1, VEGF[Alfa], CD20 and PGR genes are markers for tumourigenesis, whereas MMP3, EGF, PDL-1, and RLN2 genes are markers for malignancy. Gene expressions for CD20, PGR, EGF, RLN2 and MMP3 are significantly correlated, suggesting that the combination of these molecules may have potential as malignancy biomarkers in CMTs. In conclusion, this study demonstrated the involvement of specific serum CD proteins as well as specific genes expressed in mammary tissue in the detection of tumoural anomalies.