Genetic alterations in the fibroblast growth factor receptor (FGFR) pathway are promising therapeutic targets in many cancers, including intrahepatic cholangiocarcinoma (ICC). The FGFR inhibitor BGJ398 displayed encouraging efficacy in patients with FGFR2 fusion-positive ICC in a phase II trial, but the durability of response was limited in some patients. Here, we report the molecular basis for acquired resistance to BGJ398 in three patients via integrative genomic characterization of cell-free circulating tumor DNA (cfDNA), primary tumors, and metastases. Serial analysis of cfDNA demonstrated multiple recurrent point mutations in the kinase domain at progression. Accordingly, biopsy of post-progression lesions and rapid autopsy revealed marked inter- and intralesional heterogeneity, with different mutations in individual resistant clones. Molecular modeling and studies indicated that each mutation led to BGJ398 resistance and was surmountable by structurally distinct FGFR inhibitors. Thus, polyclonal secondary mutations represent an important clinical resistance mechanism that may guide the development of future therapeutic strategies. We report the first genetic mechanisms of clinical acquired resistance to FGFR inhibition in patients with FGFR2 fusion-positive ICC. Our findings can inform future strategies for detecting resistance mechanisms and inducing more durable remissions in ICC and in the wide variety of cancers where the FGFR pathway is being explored as a therapeutic target. .