The aim of the study was to evaluate an immunohistochemical technique for the diagnosis of canine mixed mammary tumors (CMMT) using anti-cytokeratin AE1/AE3 and anti-vimentin monoclonal antibodies that are routinely used in the diagnosis of human mammary tumors. Twenty paraffin tissue blocks of canine mixed mammary tumors, previously diagnosed by hematoxilin-eosin (HE) histopathology were used. Three slides sets, 4 μm thick, were obtained per tissue block. One was stained with HE to confirm the viability of the tissue, and the other two were used for the immunohistochemistry through the Streptavidin-Biotin-labeled method. Epithelial cells positively reacted against the anti-cytokeratin AE1/AE3 showing a brown colour at the cytoplasma level. Fibroblasts, connective tissue, myoepithelial cells, cartilage cells and bone areas showed a brown colour against the anti-vimentin antibody. All benign tumors reacted positively to both antibodies, whereas 73% malign tumors were positive with the anti-cytokeratin antibody. It is concluded that mixed mammary neoplasm in dogs may be diagnosed by the technique of immunohistochemistry using monoclonal antibodies used in the detection of human mammary tumors.