Haploidentical hematopoietic stem cell transplantation (h-HSCT) is a therapeutic option to cure patients affected by hematologic malignancies. The kinetics and the quality of immune-reconstitution (IR) impact the clinical outcome of h-HSCT and limit the onset of life-threatening Human Cytomegalovirus (HCMV) infection/reactivation. Natural Killer (NK) cells are the first lymphocytes that recover after h-HSCT and they can provide rapid innate immune responses against opportunistic pathogens. By performing a longitudinal single-cell analysis of multiparametric flow-cytometry data, we show here that the persistence at high frequencies of CD158b1b2j^neg/NKG2A^pos/NKG2C^neg/NKp30^pos/NKp46^pos (KIR^neg) NK cells is associated with HCMV infection/reactivation control. These KIR^neg NK cells are “unlicensed”, and are not terminal-differentiated lymphocytes appearing early during IR and mainly belonging to CD56^bright/CD16^neg and CD56^bright/CD16^pos subsets. KIR^neg NK cells are enriched in oxidative and glucose metabolism pathways, produce interferon-γ, and are endowed with potent antiviral activity against HCMV ex vivo. Decreased frequencies of KIR^neg NK cells early during IR are associated with clinically relevant HCMV replication. Taken together, our findings indicate that the prolonged persistence of KIR^neg NK cells after h-HSCT could serve as a biomarker to better predict HCMV infection/reactivation. This phenomenon also paves the way to optimize anti-viral immune responses by enriching post-transplant donor lymphocyte infusions with KIR^neg NK cells.