¹Henan Institute of Science and Technology, College of Animal Science and Veterinary Medicine, 453003 Xinxiang, CHINA DOI : 10.9775/kvfd.2025.34925 Biomarkers play critical roles in understanding tumor biology and evaluating prognosis in canine mammary tumor (CMTs) research. MTDH and Ki-67 are crucial factors and markers in the carcinogenesis of multiple organs and tissues in human oncology. However, the role of MTDH in CMTs and its relationship with Ki-67 are not well characterized. This study investigated MTDH and Ki-67 expression and their correlation in 64 benign and malignant CMT tissues using immunohistochemistry (IHC). The association of MTDH and Ki-67 expression with clinicopathological features was also evaluated, followed by assessing their potential prognostic value in a prospective survival study. IHC analysis revealed MTDH expression in both the cytoplasm and nucleus of tumor cells. In contrast, Ki-67 was predominantly in the nucleus. MTDH expression significantly correlated with tumor malignancy grade (P=0.035), tumor size (P<0.0001), Ki-67 index (P<0.0001), and metastasis (P<0.0001). High MTDH expression was significantly associated with reduced disease-free survival (P=0.0042) and overall survival (P=0.0113) in malignant CMTs. These results indicate that the expression levels of MTDH and Ki-67 are positively correlated with adverse clinicopathological parameters and jointly signify aggressive tumor behavior and poor prognosis. MTDH and Ki-67 are thus potential prognostic biomarkers for CMTs. Keywords : Immunohistochemistry, KI-67, Mammary Tumors, MTDH