BACKGROUND Acute myeloid leukemia with intermediate cytogenetic risk (ICR-AML) needs to be stratified. The abnormal gene expression might be prognostic, and its cutoff value for patient grouping is pivotal. MATERIAL AND METHODS Ecotropic viral integration site 1 (EVI1) transcripts were assessed in 191 adult ICR-AML patients at diagnosis who received chemotherapy only. MLL-PTD, WT1 transcript levels, FLT3-ITD, and NPM1 mutations were simultaneously evaluated, and 27 normal bone marrow samples were tested to define normal threshold. RESULTS The normal upper limit of EVI1 transcript levels was 8.0%. Receiver operating characteristic curve analysis showed that 1.0% (a 0.9-log reduction from the normal limit) was the EVI1 optimal cutoff value for significantly differentiating relapse (P=0.049). A total of 23 patients (12%) had EVI1 levels ≥1.0%. EVI1 ≥1.0% had no effect on CR achievement, whereas it was significantly associated with lower 2-year relapse-free survival (RFS), disease-free survival (DFS), and overall survival (OS) rates in the entire cohort (P=0.0003, 0.0017, and 0.0009, respectively), patients with normal karyotypes (P=0.0032, 0.0047, and 0.0007, respectively), and FLT3-ITD (-) patients (all P<0.0001). Multivariate analysis showed that EVI1 ≥1.0% was an independent adverse prognostic factor for RFS, DFS, and OS in the entire cohort. In addition, patients with EVI1 transcript levels between 1.0% and 8.0% had 2-year RFS rates similar to those with EVI1 ≥8.0%, and they both had significantly lower RFS rates than those with EVI1 <1.0% (P=0.0005 and 0.027). CONCLUSIONS High EVI1 expression predicts poor outcome in ICR-AML patients receiving chemotherapy. The optimal cutoff value for patient stratification is different from the normal limit.