We have demonstrated previously that human embryonic stem (hES) cells possess a characteristic morphologic, antigenic, and molecular profile that can be used to assess the state of ES cells (Carpenter et al., 2004 Dev Dyn 229:243–258). In this manuscript, we have examined the long‐term stability of three hES cell lines in feeder‐free culture. We demonstrate that the expression of antigens and transcription factors, telomerase activity, telomere length, and karyotype appear stable for all three hES cell lines after continuous culture for over 1 yr. All three lines retained pluripotent differentiation in vitro and in vivo. Although hES cell lines were remarkably stable over the period of analysis, a detailed quantitative analysis of antigen expression by flow cytometry and gene expression by microarray suggested that cell lines show subtle differences in the expression of small subsets of genes upon long‐term culture. Developmental Dynamics 229:259–274, 2004. © 2003 Wiley‐Liss, Inc.