Background: Cholangiocarcinoma (CCA) has limited treatment options. Genomic analyses have led to development of targeted therapies now in several clinical trials, and may enable the discovery of new treatment options. However, biopsy often yields limited tissue, thus hampering tissue-based profiling opportunities. Data regarding circulating tumor DNA (ctDNA) plasma analysis in CCA during real time clinical practice is limited. Methods: We performed ctDNA NGS analysis in patients with advanced CCA (January 2015 – December 2017). ctDNA analysis was performed using Guardant 360, which detects single nucleotide variants, amplifications, fusions, and specific insertion/deletion mutations in up to 73 different genes. Seventeen samples were performed on previous panel versions (3 on a 54-gene, 1 on a 68-gene, and 13 on a 70-gene panel) The mutant allele fraction (MAF) for detected alterations was calculated relative to wild type in ctDNA. Actionability was defined as possible treatments within OncoKB levels I-IV and R1. The study was conducted in accordance with Mayo Clinic Institutional Review Board requirements. Results: Among 104 patients and 115 total samples, ctDNA NGS revealed at least one genomic alteration (excluding variants of uncertain significance (VUS) and synonymous mutations) in 80 patients (77%). Median number of alterations per patient was 3 [range, 1-15], with a median mutant allele fraction of 0.42% (range, 0.1% - 94.2%). The total number of unique alterations was 389, with the most commonly altered genes being: TP53 (84 alterations, 22%), followed by KRAS (34 alterations, 9%), FGFR2 (31 alterations, 8%), ARID1A (20 alterations, 5%), APC and PIK3CA (16 alterations each, 4%). Amplifications were noted in 14 genes, including BRAF, CCND1, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, MET, MYC, PDGFRA, and PIK3CA. Fusions of FGFR2 were seen in 3 cases. Potentially actionable alterations were seen in 63 of the 104 patients (61%). Conclusions: ctDNA plasma profiling of patients with advanced CCA is a feasible alternative method to gather comprehensive genomic data. Further study of responses to ctDNA NGS-guided over tissue-guided targeted therapy is needed to define the best means to optimize outcomes in CCA.