Breast cancer in humans and mammary tumors in cats and dogs is one of the most important types of cancer and causes serious losses. Early diagnosis is crucial, and the treatment protocols are often complex, expensive, and inconclusive. Mast cells are considered among the important components of the immune system and have been documented to show a significant increase in cancer tissues, however their possible roles and their phenotypes in cancer are not precisely known. In this study, we examined the immunophenotypes of mast cells and their potential role in naturally occurring feline and canine mammary tumors through an experimental mammary cancer model induced by 7, 12-dimethylbenzanthracene (DMBA) in rats. The study also questioned expressions of TNF-alpha, MMP-9, and PCNA, and their possible relationship with mast cells. Mast cell count and both chymase- and tryptase-positive mast cells were increased in tumor tissues from all three species compared to the control mammary tissues. Degranulated mast cells were more common in intratumoral areas, and granulated mast cells were more common in peritumoral areas. In mammary tumors of rats, expression of PCNA correlated negatively with mast cell count; in dogs and cats, a correlation was seen, but could not be statistically substantiated. In conclusion, the increase in TNF-a, the decrease in MMP-9, and the negative correlation observed between PCNA and mast cell count indicated that an increase in mast cell count may have an anti-tumorigenic effect in mammary tumors. As a conclusion of the study, the number, localization, granulation status, and immunophenotypic characteristics of mast cells and their possible roles in mammary tumors of cats, dogs, and rats were investigated, and it is suggested that mast cells may play an important role in mammary tumors and may prove to be valuable prognostic markers.