To identify therapeutic targets in acute myeloid leukemia (AML), we chemically interrogated 200 sequenced primary specimens. Mubritinib, a known ERBB2 inhibitor, elicited strong anti-leukemic effects in vitro and in vivo. In the context of AML, mubritinib functions through ubiquinone-dependent inhibition of electron transport chain (ETC) complex I activity. Resistance to mubritinib characterized normal CD34+ hematopoietic cells and chemotherapy-sensitive AMLs, which displayed transcriptomic hallmarks of hypoxia. Conversely, sensitivity correlated with mitochondrial function-related gene expression levels and characterized a large subset of chemotherapy-resistant AMLs with oxidative phosphorylation (OXPHOS) hyperactivity. Altogether, our work thus identifies an ETC complex I inhibitor and reveals the genetic landscape of OXPHOS dependency in AML. •Mubritinib exhibits strong in vitro and in vivo anti-AML activity•It is selectively toxic against a distinct and large subset of poor outcome AMLs•Mubritinib is a ubiquinone-dependent NADH dehydrogenase inhibitor•Mubritinib-sensitive primary leukemias exhibit OXPHOS hyperactivity Through a chemical screen in sequenced primary AML specimens, Baccelli et al. identify mubritinib, a known ERBB2 inhibitor, to be selectively toxic against a large subset of poor outcome AML specimens that exhibit oxidative phosphorylation hyperactivity by inhibiting the electron transport chain complex I.