Purpose: Micrometastatic cells detected in the bone marrow have prognostic significance in breast cancer. These cells are heterogeneous and likely do not exhibit uniform biological behavior. To understand the molecular diversity of disseminated cancer cells that reside in bone marrow, we enriched this cell population and did global gene expression profiling in the context of a prospective clinical trial involving women with clinical stage II/III breast cancer undergoing neoadjuvant chemotherapy. Experimental Design: Enrichment of TACSTD1 (EpCAM) - expressing cells from bone marrow of breast cancer patients was achieved using immunomagnetic beads. Gene expression profiles were compared between enriched cell populations and whole bone marrow from 5 normal volunteers and 23 breast cancer patients after neoadjuvant chemotherapy treatment. Enriched cells from bone marrow samples of breast cancer patients before treatment or at1 year follow-up were also analyzed (total of 87 data sets). The expression of transcripts specifically detected in enriched cell populations from breast cancer patients was correlated with 1-year clinical outcome using quantitative reverse transcription-PCR in an independent cohort of bone marrow samples. Results: Analysis of EpCAM-enriched bone marrow cells revealed specific expression of a subgroup of transcripts, including the metastasis regulator, TWIST1. Most transcripts identified, including TWIST1, were not expressed in enriched populations of bone marrow from normal volunteers, suggesting that this expression profile reflects a signature of breast cancer bone marrow micrometastases that persist after chemotherapy. In an independent set of bone marrow samples obtained before any treatment, TWIST1 expression correlated with early disease relapse. Conclusions: Disseminated breast cancer cells present in bone marrow after chemotherapy possess unique transcriptional signatures. Genes whose expression is overrepresented in these cell populations, such as TWIST1, may prove to be excellent markers of early distant relapse in breast cancer patients.