Introduction: Normal karyotype in Acute Myeloid Leukemia (NK-AML) is present in 40-50% patients, commonly classified as intermediate-risk and for whom prognosis is less predictable. Other prognostic markers in NK-AML could refine disease assessment and therapeutic choices. Leukemic Stem Cells (LSCs) have been associated with initiation and persistence of AML. In this study, we aimed to assess the prognostic impact of the proportion of LSCs at diagnosis. Methods: Patients with de novo NK-AML (n = 54,19-71 years old) from 7 Brazilian centers enrolled in the International Consortium of Acute Leukemias Study–IC-AML2015 had bone marrow samples taken to assessment of FLT3 and NPM1 mutations, RUNX1/RUNX1T1 and CBFB-MYH11 rearrangements, karyotype analysis by classical cytogenetics and immunophenotyping by multiparametric flow cytometry. Data regarding ELN2017-based risk stratification (without TP53, ASXL1 and RUNX1) and overall survival (OS) were collected. Treatment comprised two cycles of induction, and consolidation with one or two cycles of chemotherapy and/or BM transplantation, according to clinical appraisal. LSCs were evaluated according to the percentage of CD34+/CD38low/CD123+ cells among total blast cells (from SSC versus CD45 gating population) and to their absolute numbers. Analyses were performed with SPSS (V.20), considering a p-value of 0.05. Results: Patients were classified in two groups (LSC<1%; LSC>1%). The presence of LSC>1% was associated with higher WBC (49.2 x 103/μL x 13.6 x 103/μL, p = 0.02), with combined NMP1 and FLT3-ITD mutations (88.9% of patients x 11.1%, p < 0,001) and with high FLT3-ITD allelic ratio (44.4% x 4.4%, p < 0,001). Complete remission rate 69% (n = 31) among the LSC<1% group, and 34% (n = 3) among the LSC>1% group. Within the whole cohort, those with LSC >1% (n = 9) had a mean OS of 6.3 months, while a 24.2 months OS was observed in patients of the LSC <1% group (n = 45). Among intermediate-risk patients, those with LSC >1% (n = 6) had a mean OS of 7.1 months, whereas the LSC <1% group (n = 25) had a 23.9 months OS. The logrank test demonstrated, however, equality of survival distributions between the patients with LSC <1% or LSC >1% within the total cohort and within each risk group. Discussion: The increased understanding of AML pathogenesis has prompted interest in LSC as possible prognostic markers and therapeutic targets. In our cohort, higher percentages of LSCs (>1%) at diagnosis were associated with markers of inferior prognosis: higher leukocytosis, NPM1mutFLT3mut status and higher FLT3-ITD allelic ratio. In the group of intermediate-risk, those with LSC>1% presented a lower mean overall survival than that of the patients with LSC <1% at diagnosis. This finding is relevant for this group of patients, for which prognosis and therapy choices are not as well defined as for the low and high-risk patients. However, logrank test displayed equality of survival distributions between the two groups of LSC, which could have been due to the limited number of patients studied. Conclusion: Our results fortify the potential value of LSCs as an easily assessed prognostic factor at diagnosis that may be further evaluated along measurable residual disease time points and help on therapeutic decisions. Funding: FAPESP grant #2013/08135-2. GLJ: FAPESP grant #19/20215-8. AFOC: INCTC grant #88887.284979/2018-00. LOM: INCTC grant #88887.313021/2019-00. LLFP: FAPESP grant #2015/21866-1.