Updated publication reference for PubMed record(s): 30530751. Anti-leukemia immunity plays an important role for disease control and maintenance of tyrosine kinase inhibitor (TKI)-free remission in chronic myeloid leukemia (CML). Antigen-specific immunotherapy holds promise to strengthen immune control in CML, but requires the identification of CML-associated targets. In this study, we used a mass spectrometry-based approach to identify naturally presented HLA class I- and class II-presented peptides in 20 primary CML samples. Comparative HLA ligandome profiling using a comprehensive dataset of different hematological benign specimen delineated a novel panel of frequently expressed CML-exclusive peptides. These non-mutated target antigens are of particular relevance since our extensive data-mining approach demonstrated absence of naturally presented BCR-ABL- and ABL-BCR-derived HLA-presented peptides and the lack of frequent tumor-exclusive presentation of predescribed cancer/testis and leukemia-associated antigens. Functional characterization revealed spontaneous T-cell responses against the newly identified CML-associated peptides in CML patients and their ability to de novo induce multifunctional and cytotoxic antigen-specific T cells in healthy volunteers and CML patients. This characterizes these antigens as prime candidates for T cell-based immunotherapy approaches that may prolong TKI-free survival and even mediate cure of CML patients.