CD56neg CD16+ Natural Killer (NK) cells have been reported to expand in chronic diseases and acute myeloid leukemia (AML). However, their biological role is still unclear. Using mass cytometry, spectral flow cytometry, bulk RNA-seq and in vitro assays, we characterized CD56neg CD16+ NK cells that expand in AML. We confirmed that CD56neg CD16+ NK cells represent a unique NK cell subset coexpressing Eomes and T-bet. CD56neg CD16+ NK cells could recover CD56 expression in vitro where they displayed unaltered NK cell functions. We previously demonstrated that CD56neg CD16+ NK cells expansion at diagnosis was associated with adverse clinical outcome in AML. Here, we validated our findings in a validation cohort of N=38 AML patients. AML patients with CD56neg CD16+ NK cells expansion at diagnosis had decreased overall survival (HR[CI95]=5.5[1.2-24.5], p=0.0251) and relapse-free survival (HR[CI95]=13.1[1.9-87.5], p=0.0079) compared to AML patients without expansion after 36 months follow-up. RNA-seq unveiled that CD56neg CD16+ NK cells were mature circulating NK cells with functional capacities. Upon expansion, CD56neg CD16+ NK cells from AML patients showed altered proteomic phenotype, with high expression of TIM-3 and Siglec-7. Taken together, our results suggest that CD56neg CD16+ NK cells are a relevant target for future NK-cell-based immunotherapies.