The cancer stem cell (CSC) hypothesis proposes that tumour growth is maintained by a distinct subpopulation of CSC. This study applied flow cytometric methods, reported to detect CSC in both primary and cultured cancer cells of other species, to identify candidate canine subpopulations. Cell lines representing diverse canine malignancies, and cells derived from spontaneous canine tumours, were evaluated for expression of stem cell-associated surface markers (CD34, CD44, CD117 and CD133) and functional properties [Hoecsht 33342 efflux, aldehyde dehydrogenase (ALDH) activity]. No discrete marker-defined subsets were identified within established cell lines; cells derived directly from spontaneous tumours demonstrated more heterogeneity, although this diminished upon in vitro culture. Functional assays produced variable results, suggesting context-dependency. Flow cytometric methods may be adopted to identify putative canine CSC. Whilst cell lines are valuable in assay development, primary cells may provide a more rewarding model for studying tumour heterogeneity in the context of CSC. However, it will be essential to fully characterize any candidate subpopulations to ensure that they meet CSC criteria.