ABSTRACTMolecular mechanisms behind potentially inferior prognosis of old cholangiocarcinoma (CCA) patients are unclear. Prevalence of interventional targets and the difference between young and old CCA patients are valuable for promising precision medicine. A total of 188 CCA patients with baseline tumor tissue samples were subgrouped into the young (≤45 years) and old (>45 years) sub-cohorts. Somatic and germline mutation profiles, differentially enriched genetic alterations, and actionable genetic alterations were compared. An external dataset was used for the validation of molecular features and the comparison of overall survival (OS). Compared to young patients,KRASalterations were more common in old patients (P= .04), whileFGFR2fusions were less frequent (P= .05).TERTpromoter mutations were exclusively detected in old patients. The external dataset (N= 392) revealed no significant difference in OS between young and old patients; however, old patient-enrichedKRAS(hazard ratio [HR]: 1.96, 95% confidence interval [CI]: 1.37–2.80) andTERTalterations (HR: 2.03, 95% CI: 1.22–3.38) were associated with inferior OS. Approximately 38.3% of patients were identified of actionable oncogenic mutations indicative of a potential response to targeted therapy or immunotherapy. ActionableFGFR2fusions (P= .01) andBRAFV600E(P= .04) mutations were more frequent in young females than old patients. The enrichment ofKRAS/TERTalterations in CCA patients over 45 years resulted in inferior OS. Approximately one-third of CCA patients were eligible for targeted therapy or immunotherapy given the actionable mutations carried, especially young females.