Mammary neoplasms affect a population of intact and elderly female dogs and 50% of these neoplasms are malignant. In order to study this disease, cell culture is as a promising preclinical model, creating the opportunity to deposit cell lines at a cell bank, allowing a great reproducibility of the assays and making the validation of the results more reliable. Another important aspect is the possibility to establish models for the better understanding of tumour characteristics, such as vasculogenic mimicry. VM is related to cancer cells able to generate vascular-like structures from epithelial cells, mimicking the vasculogenic process and it is closely related to patient’s prognosis. Due the importance of cancer cell lines in preclinical models, this study aimed to establish and characterize ten cell lines from canine mammary gland tumours according to immunophenotype and tumorigenicity, and with its ability to form vasculogenic mimicry-like structures in vitro and in vivo. Cell cultures were evaluated for morphology, phenotype, vasculogenic mimicry and tumorigenicity abilities. All cell lines presented spindle shape morphology and expressed concomitant pan-cytokeratin and cytokeratin 8/18. Four cell lines had vasculogenic mimicry ability in vitro and two of them showed in vivo tumorigenic potential and forming VM in the xenotransplant tumour. Cell characterization of those lines will help to create a database for more knowledge of mammary carcinomas in dogs, including studies of tumor behavior and new therapeutic targets.