Background: Natural Killer (NK) cells have a role in antitumor activity, but its potential for regulating acute myeloid leukemia (AML) is not well understood. We believe its numerical or defective maturation may result in leukemic stem cell (LSC) resistance in AML. So, we aimed to immunophenotypically characterize the frequency, subtypes, and maturation of NK cells in AML samples with known prognostic factors. Methods: Bone marrow (BM) samples from 73 patients with AML from 7 Brazilian centers were assessed for FLT3 and NPM1 mutations, RUNX1/RUNX1T1 and CBFB-MYH11 gene fusions, karyotyping by classical cytogenetics and immunophenotyping by flow cytometry, using 5 normal BM as controls. NK subtypes were defined as CD19-CD3- and: CD56brightCD16- (secretory) or CD56dimCD16+ (cytotoxic). CD19-CD3-CD56+ NK cells were classified as: CD11b-CD27- (tolerant); CD27+CD11b- (immature secretory); CD27+CD11b+ (secretory) and CD11b+CD27- (cytotoxic). LSC (CD34+CD38lowCD123+) were assessed among total blast cells. Clinical, laboratorial data and ELN2017-based risk stratification were evaluated. Mann Whitney or Kruskal-Wallis tests (P < 0.05) were applied using SPSS V.20. Results: A tendency of lower lymphocyte frequency in total BM cells in AML patients was observed (p = 0.1507), but no difference in NK cell numbers or subtypes were noted. Favorable risk patients showed a higher frequency of total NK cells (p = 0.042) as well as CD56brightCD16- (p = 0.0051) and CD56dimCD16+ (p = 0.072) than other risk categories or controls (p = 0.0053). Favorable risk group showed lower percentages of CD27-CD11b- and higher percentages of CD27-CD11b+ NK cells (p = 0.0401 and p = 0.0545). Normal BM displayed lower frequency of CD27-CD11b- and higher expression of CD27+CD11b- (P = 0.2591) and CD27+CD11b+ NK cells (p = 0.0106) than AML samples. In normal or abnormal karyotype groups the frequency of total CD56/CD16 NK subtypes was not significant, but increased total NK numbers were found in normal karyotype patients (p = 0.1994). Of note, CD27CD11b- NK cells were mostly found in abnormal karyotype samples (p = 0.0984), and a higher frequency of CD27-CD11b+ NK cells was noted in normal karyotype samples (p = 0.0285). Patients with FLT3-ITD or NPM1 mutations showed lower numbers of CD56dimCD16+ (p = 0.0388 and p = 0.0364). In addition, total NK (p = 0.0159) and both CD56brightCD16- (p = 0.0386) or CD56dimCD16+ (p = 0.0337) frequency was higher in CBFB-MYH11 leukemia. Lastly, an intermediate correlation between LSC quantification and CD27+CD11b- NK cells was found (R = 0.429, p < 0.01). Conclusion: Phenotypic changes in NK cells may be related to risk stratification in AML. The higher frequency of NK cells and its cytotoxic subtypes in favorable-risk patients suggests that activated NK cells may contribute to better outcomes. Patients without karyotype abnormalities or FLT3 -ITD mutation also showed higher expression of cytotoxic cells, what could be confirmed in CBFB-MYH11 leukemia, which presented higher counts of NK cells and its subtypes. In contrast, abnormal karyotype and more adverse-risk patients showed higher numbers of immature non-effective NK cells. The correlation between LSC quantification and increased immature secretory NK cells leads us to believe that numerical and/or functional NK impairment may contribute to AML maintenance. Conclusions regarding NK profile, treatment response, and long-term outcomes need to be determined to further justify immunotherapy mediated by NK cells.