DNA microarrays have the potential to classify tumors according to their transcriptome. Tissue microarrays (TMAs) facilitate the validation of biomarkers by offering a high-throughput approach to sample analysis. We reanalyzed a high profile breast cancer DNA microarray dataset containing 96 tumor samples using a powerful statistical approach, between group analyses. Among the genes we identified was centromere protein-F (CENP-F), a gene associated with poor prognosis. In a published follow-up breast cancer DNA microarray study, comprising 295 tumour samples, we found that CENP-F upregulation was significantly associated with worse overall survival (p < 0.001) and reduced metastasis-free survival (p < 0.001). To validate and expand upon these findings, we used 2 independent breast cancer patient cohorts represented on TMAs. CENP-F protein expression was evaluated by immunohistochemistry in 91 primary breast cancer samples from cohort I and 289 samples from cohort II. CENP-F correlated with markers of aggressive tumor behavior including ER negativity and high tumor grade. In cohort I, CENP-F was significantly associated with markers of CIN including cyclin E, increased telomerase activity, c-Myc amplification and aneuploidy. In cohort II, CENP-F correlated with VEGFR2, phosphorylated Ets-2 and Ki67, and in multivariate analysis, was an independent predictor of worse breast cancer-specific survival (p = 0.036) and overall survival (p = 0.040). In conclusion, we identified CENP-F as a biomarker associated with poor outcome in breast cancer and showed several novel associations of biological significance. © 2006 Wiley-Liss, Inc.