Mammaglobin-A (MAM-A) is a glycoprotein predominantly linked to mammary tissue. It is a secretory protein, characterized by its composition of 93 amino acids and a molecular weight of around 10 kDa. The primary objective of this study is to evaluate the tissue expression of MAM-A in different histotypes of canine mammary tumors (CMT). The present study includes a total of 40 samples of CMT and 5 samples of apparently healthy canine mammary tissues, which were subjected to histological and immunohistochemical examinations. Hyperimmune serum was generated against recombinant MAM-A (rMAM-A) in rabbits to establish a standardized immunohistochemical protocol for evaluating the expression of MAM-A protein in different histotypes of CMT. The histological study demonstrated a wide array of tumor classifications, predominantly consisting of carcinoma, followed by carcinosarcoma and sarcoma. The immunohistochemical examination demonstrated a pronounced expression of MAM-A in the majority of CMT histotypes. The immunohistochemical expression of MAM-A in CMT indicates its potential as a valuable diagnostic biomarker.