BACKGROUND & AIMS: Brain metastases (BMs) from colorectal cancer (CRC) are associated with significant morbidity and mortality, with chemoresistance and short overall survival. Migrating cancer stem cells with the ability to initiate BM have been described in breast and lung cancers. In this study, we describe the identification and characterization of cancer stem cells in BM from CRC. METHODS: Four brain metastasis stem cell lines from patients with colorectal cancer (BM-SC-CRC1 to BM-SC-CRC4) were obtained by mechanical dissociation of patient's tumors and selection of cancer stem cells by appropriate culture conditions. BM-SC-CRCs were characterized in vitro by clonogenic and limiting-dilution assays, as well as immunofluorescence and Western blot analyses. In ovo, a chicken chorioallantoic membrane (CAM) model and in vivo, xenograft experiments using BALB/c-nude mice were realized. Finally, a whole exome and RNA sequencing analyses were performed. RESULTS: BM-SC-CRC formed metaspheres and contained tumor-initiating cells with self-renewal properties. They expressed stem cell surface markers (CD44v6, CD44, and EpCAM) in serum-free medium and CRC markers (CK19, CK20 and CDX-2) in fetal bovine serum-enriched medium. The CAM model demonstrated their invasive and migratory capabilities. Moreover, mice intracranial xenotransplantation of BM-SC-CRCs adequately recapitulated the original patient BM phenotype. Finally, transcriptomic and genomic approaches showed a significant enrichment of invasiveness and specific stemness signatures and highlighted KMT2C as a potential candidate gene to potentially identify high-risk CRC patients. CONCLUSIONS: This original study represents the first step in CRC BM initiation and progression comprehension, and further investigation could open the way to new therapeutics avenues to improve patient prognosis.