Objective: Bone-marrow mesenchymal stem cells (MSCs) are multipotent cells capable of self-renewal and differentiation into multiple cell types. Accumulated preclinical and clinical evidences indicate that MSCs are good candidates to be used in cell therapy for the treatment of many degenerative diseases. For MSCs clinical applications, an adequate number of cells are necessary so an extensive in vitro expansion is required. The importance of analyzing rat MSCs (rMSCs) is related to their use as a model for understanding their human MSCs (hMSCs) relatives. Materials and Methods: We evaluated and compared the biological characteristics and the chromosomal status of rMSCs and hMSCs at several culture passages in vitro. We first used the conventional traditional cytogenetic techniques, in order to have the opportunity to observe even minor structural abnormalities and to identify low-degree mosaic conditions. Subsequently, a more detailed genomic analysis was conducted by array comparative genomic hybridization. Results: We demonstrated that, rMSCs manifested a markedly aneuploid karyotype and a progressive chromosomal instability in all the passages we analyzed and that they are anything but stable during in vitro culture. On the contrary, hMSCs in vitro cultures were characterized by a normal karyotype and chromosomal stability in most of the donors and culture passages examined. Conclusion: Our results support the idea that there is a considerable risk for long-term rMSC cultures due to their genomic instability. Despite the fact that the risk of neoplastic transformation associated with this genomic instability needs to be further addressed and considering the apparent genomic stability reported for in vitro cultured hMSCs, our findings underline the fact that rMSCs may not in fact be a good model for effectively exploring the full clinical therapeutic potential of hMSCs.