Mesenchymal stem cells (MSCs) of human and nonhuman mammalian species are often studied for various applications in regenerative medicine research. These MSCs can be derived from human bone marrow (BM) and identified by their ability to form fibroblast-like colony forming units that develop into stromal like cells when expanded in culture. These cells are characterized by their spindle-shaped morphology, their characteristic phenotype (CD73(+), CD90(+), CD105(+), CD45⁻, and CD34⁻), and their ability to differentiate into cells of the osteogenic, adipogenic, and chondrogenic lineages. However, the identification and purification of MSCs from nonhuman mammalian species is hampered by the lack of suitable monoclonal antibodies (mAb). In this report, primary BM and cultured BM-derived MSCs of human and monkey, goat, sheep, dog, and pig were screened for cross-reactivity using a panel of 43 mAb, of which 22 react with either human BM mononuclear cells or cultured human MSCs. We found 7 mAb with specificity for CD271, MSCA-1 (W8B2 antigen), W4A5, CD56, W3C4 (CD349), W5C4, and 58B1, which showed interspecies cross-reactivity. These mAb proved to be useful for prospective sorting of MSCs from the BM of the 6 mammalian species studied as well as for the characterization of their cultured offspring. Flow sorting with the cross-reacting mAb resulted in up to 2400-fold enrichment of the clonogenic cell fraction (fibroblast-like colony forming units). This study provides an important contribution for the comparative prospective isolation of primary BM-MSCs and the characterization of cultured MSCs from multiple mammalian species for preclinical research.