Abstract Oncogenic activation of the FGFR pathway is frequent in lung and other cancers. However, due to drug resistance, pharmacological blockage of aberrant FGFR signaling has provided little clinical benefit in patients with FGFR-amplified tumors. The determining factors for the limited efficacy of FGFR-targeted therapy remain incompletely understood. In this study, we performed kinome-wide CRISPR/Cas9 loss-of-function screens in FGFR1-amplified lung cancer cells treated with an FGFR inhibitor. These screens identified PLK1 as a potent synthetic lethal target that mediates a resistance mechanism by overriding DNA damage and cell-cycle arrest upon FGFR1 inhibition. Genetic and pharmacological antagonism of PLK1 in combination with FGFR inhibitor therapy synergized to enhance antiproliferative effects and drove cancer cell death in vitro and in vivo through activation of the γH2AX–CHK–E2F1 axis. These findings suggest a previously unappreciated role for PLK1 in modulating FGFR1 inhibitor sensitivity and demonstrate a synergistic drug combination for treating FGFR1-amplified lung cancer. Significance: The identification of PLK1 as a potent synthetic lethal target for FGFR-targeted therapy provides an innovative rationale for the treatment of lung and other FGFR1-amplified cancers.