The proto-oncogene EVI1 encodes a DNA binding protein and is located on chromosome 3q26. The gene is aberrantly expressed in acute myeloid leukemia (AML) patients carrying 3q26 abnormalities. Two mRNAs are transcribed from this locus: EVI1 and a fusion ofEVI1 with MDS1(MDS1-EVI1), a gene located 5′ ofEVI1. The purpose of this study was to investigate which of the 2 gene products is involved in transformation in human AML. To discriminate between EVI1 andMDS1-EVI1 transcripts, distinct real-time quantitative polymerase chain reaction (PCR) assays were developed. Patients with 3q26 abnormalities often showed highEVI1 and MDS1-EVI1 expression. In a cohort of 319 AML patients, 4 subgroups could be distinguished:EVI1⁺ andMDS1-EVI1⁻ (6 patients; group I), EVI1⁺ andMDS1-EVI1⁺ (26 patients; group II),EVI1⁻ andMDS1-EVI1⁺ (12 patients; group III), and EVI1⁻ andMDS1-EVI1⁻ (275 patients; group IV). The only 4 patients with a 3q26 aberration belonged to groups I and II. Interestingly, high EVI1 and notMDS1-EVI1 expression was associated with unfavorable karyotypes (eg, −7/7q−) or complex karyotypes. Moreover, a significant correlation was observed betweenEVI1 expression and 11q23 aberrations (mixed lineage leukemia [MLL] gene involvement). Patients from groups I and II had significantly shorter overall and event-free survival than patients in groups III and IV. Our data demonstrate that highEVI1 expression is an independent poor prognostic marker within the intermediate- risk karyotypic group.