Abstract Background: Biliary tract cancer (BTC) is an aggressive malignancy associated with a poor prognosis. Most BTC patients are diagnosed at an unresectable stage; further, the efficacy of cytotoxic chemotherapy for BTC remains unsatisfactory. Recently, there is growing evidence that gut microbial dysbiosis is related to multiple diseases, including biliary disease and cancer. However, the association between tumor and gut microbiome environments and BTC, especially extrahepatic cholangiocarcinoma (EH-CCA), remains unclear. In the present study, we compared the microbiome from the tissue and stool samples of EH-CCA patients and healthy living liver donors (controls). Methods: A total of 24 people (13 EH-CCA patients and 11 healthy controls) were included. We collected fecal and swab samples, which were obtained from the EH-CCA tissues of the patients and normal gallbladder tissues of the controls during operation. The bacterial 16S rRNA gene (V3 and V4 region) was amplified using PCR and sequenced on the Illumina MiSeq platform. The QIIME 2 pipelines were used to analyze the raw data. Results: The median age of the patients and controls were 70 (range, 58-79) and 29 (range, 19-35) years, respectively. The swab samples from EH-CCA patients showed significantly lower alpha diversity than the samples from the controls (pShannon < 0.001). Proteobacteria was the most commonly found phylum (53%) in the EH-CCA patients; Firmicutes (79%) and Actinobacteria (13%) were the common phyla in the controls. The genera Streptococcus (25.1%), Escherichia_Shigella (16.9%), and Enterococcus (13.9%) were found in the patient samples, whereas Blautia (12%), Limosilactobacillus (12%), and Bifidobacterium (19%) were observed in the controls. The Principal Coordinate Analysis (PCoA) plot of the weighted UniFrac distances showed that the patient and control groups formed separate clusters. Among the fecal samples, Streptococcus (12.5%), bacterioides (8.2%) is most common in patients’ group, whereas Blautia (13.8%) and Faecalibacterium (11.5%) is most common in control group at the gene levels. The PCoA plot of the fecal samples showed significant differences in microbiota composition between the EH-CCA patients and healthy controls (P=0.001 by PERMANOVA). In the beta diversity analysis of all samples (swab plus fecal), the patient-swab samples formed distinct clusters, whereas the patient-fecal, control-swab, and control-fecal samples showed overlapping clusters.Conclusions: To the best of our knowledge, this study is the first that compares the microbiome of tissues and stools from EH-CCA patients and healthy controls; the EH-CCA patients and healthy controls showed different microbiota profiles. Further studies are needed to investigate the clinical implications of microbial dysbiosis in BTC and formulate novel microbiome-based treatments for BTC. Citation Format: Sora Kang, Jihee Kang, Gun-Seok Park, Seung-Hyun Ko, Jung-Hyun Park, Yunseon Jang, Ik-Chan Song, Hyewon Ryu, Seok-Hwan Kim. Comparison of the tissue and gut microbiome in extrahepatic cholangiocarcinoma patients and healthy liver donors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5903.