In this study, we provide comprehensive analyses of mesenchymal stem cells (MSCs) isolated from three types of canine tissues: bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and amniotic tissue (AM-MSCs). We compare their morphology, phenotype, multilineage potential and proliferation activity. The BM-MSCs and AM-MSCs showed fibroblast-like shapes against the spindle shape of the AT-MSCs. All populations showed strong osteogenic and chondrogenic potential. However, we observed phenotypic differences. The BM-MSCs and AT-MSCs revealed high expression of CD29, CD44, CD90 and CD105 positivity compared to the AM-MSCs, which showed reduced expression of all the analysed CD markers. Similarly, the isolation yield and proliferation varied depending on the source. The highest isolation yield and proliferation were detected in the population of AT-MSCs, while the AM-MSCs showed a high yield of cells, but the lowest proliferation activity, in contrast to the BM-MSCs which had the lowest isolation yield. Thus, the present data provide assumptions for obtaining a homogeneous MSC derived from all three canine tissues for possible applications in veterinary regenerative medicine, while the origin of isolated MSCs must always be taken into account.