Therapies employing chimeric antigen receptor T cells (CAR-T cells) targeting tumour-associated antigens (TAAs) can lead to on-target–off-tumour toxicity and to resistance, owing to TAA expression in normal tissues and to TAA expression loss in tumour cells. These drawbacks can be circumvented by CAR-T cells targeting tumour-specific driver gene mutations, such as the four-nucleotide duplication in the oncogene nucleophosmin (NPM1c), which creates a neoepitope presented by the human leukocyte antigen with the A2 serotype (HLA-A2) that has been observed in about 35% of patients with acute myeloid leukaemia (AML). Here, we report a human single-chain variable fragment (scFv), identified via yeast surface display, that specifically binds to the NPM1c epitope–HLA-A2 complex but not to HLA-A2 or to HLA-A2 loaded with control peptides. In vitro and in mice, CAR-T cells with the scFv exhibit potent cytotoxicity against NPM1c⁺HLA-A2⁺ leukaemia cells and primary AML blasts, but not NPM1c^–HLA-A2⁺ leukaemia cells or HLA-A2^– tumour cells. Therapies using NPM1c CAR-T cells for the treatment of NPM1c⁺HLA-A2⁺ AML may limit on-target–off-tumour toxicity and tumour resistance.