Phenotypic and molecular parallels between the development of chondrosarcoma and the differentiation of chondrocytes in normal growth plate suggest that chondrosarcoma may arise from mesenchymal precursor cells driven towards chondrogenesis. We hypothesized that a comparison between cartilaginous tumours and their possible physiological cells of origin, mesenchymal stem cells (MSCs), might have biological and clinical relevance. MSCs from eight donors were submitted to chondrogenic differentiation in spheroid cultures. Expression profiles of MSCs at days 0, 7, 14, 28 and 42 of chondrogenesis and of 18 chondrosarcomas with different histological grades were studied using a customized cDNA array. Hierarchical clustering of MSC gene expression during chondrogenesis allowed the classification of samples in a pre-chondrogenic and a chondrogenic cluster corresponding to the phenotypes of early and late differentiation stages. The 74 genes differentially expressed between the two clusters were defined as chondrogenesis-relevant genes. Gene expression profiles of chondrosarcoma were submitted to hierarchical clustering on the basis of these chondrogenesis-relevant genes. This analysis allowed clear distinction between grade I and grade III chondrosarcoma and separated grade II chondrosarcoma into two groups. All grade II chondrosarcomas with occurrence of metastasis were found together with the grade III chondrosarcomas in the pre-chondrogenic cluster. This analysis shows that a molecular approach based on the comparison of tumour samples to an in vitro model for chondrogenic differentiation allows a new classification of chondrosarcoma in two clusters. These data suggest that the identification of a pre-chondrogenic and a chondrogenic phenotype for chondrosarcoma by gene expression profiling could develop into a useful tool to predict the clinical behaviour of chondrosarcoma. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.