Mammary neoplasms affect a population of uncastrated and elderly female dogs and most of these neoplasms are malignant. In order to study this disease cell culture presents itself as a promising preclinical model, creating the opportunity to deposit cell lines at a cell bank, allowing a great repetition of the assays and making the validation of the results more reliable. Including, in vitro experiments for vasculogenic mimicry (VM) evaluation. VM is related to cancer cells capable of generate vascular-like structures without endothelial cells, mimicking the vasculogenic process. The aim of this study was to establish and characterize ten cell lines from canine mammary gland tumour according to immunophenotype and tumorigenicity, and with its ability to form vasculogenic mimicry-like structures in vitro. Fifteen samples from canine mammary gland carcinoma were collected and cultured in vitro and ten cell lines were established and characterized. Cells were evaluated for morphology, phenotype, vascular mimicry and tumorigenicity. All cell lines presented spindle shape morphology and expressed concomitant pan-cytokeratin and CK8/18. Four cell lines had vasculogenic mimicry ability and two cell showed in vivo tumorigenic potential. Cell characterization of those lines will help to create a database for more knowledge of mammary gland carcinomas in dogs, including studies of tumor behavior and new therapeutic targets.