The present investigation was carried out with an aim to validate two commercially available Ki67 antibodies (SP6 and MIB-5) for the detection of Ki67 protein in the canine tissues, using immunohistochemical analysis. Tissues obtained from normal uterus and mammary tumor was stained with both the antibodies at the same time and following same protocol. Microscopic evaluation of stained slides showed expected nuclear pattern of Ki-67 staining with SP6 antibody in proliferative cells present at the uterine epithelium (both surface and glandular) and cancer cells of the mammary tumor. However, MIB-5antibody showed intense cytoplasmic staining only in the glandular uterine epithelial cells and very faint cytoplasmic staining in the mammary cancer cells. The results suggest that SP6 antibody has specific cross reactivity towards canine Ki67 protein and have potential usefulness in canine histopathological analysis.