Abstract Three canine mammary tumor (CMT) cell lines, namely DE-E, DE-F and DE-SF, have been established from a surgically excised specimen of a malignant mammary tumor. These CMT cell lines have been cultured for over 200 passages. The cell doubling time was estimated to be approximately 30 h for all three cell lines. DE-E, DE-F and DE-SF were epithelial, fibroblast and spindle fibroblast in morphology, respectively. Under electron microscope, DE-F and DE-SF cells displayed a higher nucleus/cytoplasm ratio as compared with DE-E. Variation in chromosome number was also observed in the three cell lines. In addition to the morphological characteristics, these cell lines displayed differential patterns of several known mammary tumor cell markers. Following xenotransplantation of the CMT cells into nude mice, DE-F and DE-SF developed tumors within 2 weeks, whereas DE-E failed to develop any visible tumor up to 8 weeks after injection. Lastly, the CMT cell lines exhibited differential chemoresistance to several anti-tumor drugs, including melatonin, cyclosporine A, tamoxifen and indole, suggesting that these cell lines can be used as a comparative experimental model for the tumorigenesis of mammary carcinomas and a valuable tool for anti-cancer drug screening.