Introduction: Chronic myeloid leukemia (CML) is characterized by the presence of the Philadelphia chromosome (Ph), resulting from a translocation between chromosomes 9 and 22, which generates the BCR-ABL1 fusion gene. Tyrosine kinase inhibitors have revolutionized the treatment of CML, but hematopoietic stem cell transplantation remains the only curative treatment, albeit with several limitations. In this context, the therapeutic use of autologous Natural Killer (NK) cells could emerge as a promising alternative if the cells are benign and maintain preserved cytotoxic activity. Objective: To assess whether NK cells from newly diagnosed CML patients are benign and possess expansion and cytotoxic capacities. Methodology: Peripheral blood samples from three healthy donors and three newly diagnosed CML patients (BCR-ABL1 positive) were depleted of T cells and expanded in vitro using a technological platform for expansion with artificial antigen-presenting cells. NK cells were isolated through Cell Sorting and analyzed for the presence of the BCR-ABL1 transcript by RT-PCR. Additionally, the cells were evaluated for their expansion capacity, cytotoxicity, and immunophenotypic profile, comparing the two groups. Results: NK cells from CML patients did not exhibit the BCR-ABL1 transcript. All cultures exhibited adequate and clinically significant expansion. In spite of expansion in the presence of modified K562 feeder cells, BCR-ABL1 negative NK cells from CML patients apparently failed to recover cytotoxic activity. Conclusion: In our study, NK cells from newly diagnosed CML patients were negative for BCR-ABL1 mutation. Nevertheless, these cells exhibited impaired cytotoxic activity even after successful expansion.