Recent data suggest that mammary carcinogenesis may be driven by cancer stem cells (CSCs) derived from mutated adult stem cells, which have acquired aberrant cell self-renewal or by progenitor cells that have acquired the capacity for cell self-renewal. Spontaneous mammary cancers in cats and dogs are important models for the understanding of human breast cancer and may represent alternative species model systems that can significantly contribute to the study of human oncogenesis. With the goal of identifying markers for isolating human breast CSCs, we have generated a canine model system to isolate and characterize normal and CSCs from dog mammary gland. Insight into the hierarchical organization of canine tumours may contribute to the development of universal concepts in oncogenesis by CSCs. Cells with stem cell properties were isolated from normal and tumoural canine breast tissue and propagated as mammospheres and tumourspheres in long-term non-adherent culture conditions. We showed that cells obtained from spheres that display self-renewing properties, have multi-lineage differentiation potential, could generate complex branched tubular structures in vitro and form tumours in NOD/SCID mice. We analysed these cells for the expression of human stem and CSC markers and are currently investigating the tumour-initiating properties of these cells and the hierarchical organization of normal and neoplastic canine mammary tissue.