Background: Fibroblast growth factor receptor 2 (FGFR2) alterations occur in 11% of cholangiocarcinomas, 85% of which are fusions. A multicenter, open-label, phase II study is currently evaluating the efficacy of infigratinib, a selective FGFR1–3 tyrosine kinase inhibitor, in patients with previously treated advanced cholangiocarcinoma containing FGFR2 fusions. We report detailed biomarker analyses from this study. Methods: Patients with advanced or metastatic cholangiocarcinoma containing FGFR2 fusions whose disease had progressed following cisplatin- or gemcitabine-based therapy were eligible. Patients received oral infigratinib 125 mg once daily on days 1–21 every 28 days. Comprehensive genomic profiling (CGP) was performed on tumor tissue and cfDNA collected prior to the start of therapy. The primary endpoint was investigator-assessed overall response rate (ORR) [RECIST version 1.1]. Data cut-off (prespecified): August 8, 2018. Trial registration: NCT02150967. Results: At data cut-off, 71 patients with FGFR2 fusions were included (62% women; median age 53 years; 55% received ≥2 prior lines of therapy). Median duration of treatment was 5.5 months. ORR (confirmed and unconfirmed) was 31.0% (95% CI 20.5–43.1%) and confirmed ORR was 26.9% (95% CI 16.8–39.1%). 33 unique FGFR2 fusion genes were identified in 71 enrolled patients. The most common fusion gene partner was BICC1 (32%; 23/71). Pathogenic variants in 9 other druggable genes were identified in 32% of patients (13/37) who underwent CGP. FGFR2 fusions were concordant in 67% (8/12) of patients with tumor tissue and cfDNA at screening. Conclusions: The large assortment of FGFR2 fusion genes identified in this study underscores the diversity of FGFR2 rearrangements that may drive cholangiocarcinoma. Although cfDNA analysis was performed in a minority, these preliminary data suggest that cfDNA analysis may be valuable for the identification of FGFR2 fusions and to study intratumoral heterogeneity. Clinical trial information: NCT02150967