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Objective: to analyze microbiota profiles in the biliary tract, of pancreatic ductal adenocarcinoma (PDAC) patients and gallstones patients, in order to identify differences, which may contribute to a better understanding of PDAC carcinogenesis. Methods: using microbiota analysis, a total of 25 samples from 14 patients were collected during surgery and compared. Samples were divided into three groups; one GS group (N = 3), and two PDAC groups; PDAC gallbladder group (N = 11) and PDAC brush group (N = 11). Results: upon comparison of bacterial communities’ alpha and beta diversity indices and relative abundances by group (anatomic site) and condition (GS vs PDAC), we found no statistically significant results. However, we can highlight the high similarity of the compared parameters among the two different anatomic locations over the biliary tract in PDAC patients. Conclusion: to the best of our knowledge, this is the first study comparing two different anatomic locations over the biliary tract in PDAC patients. Among PDAC groups microbiota along the semi-closed duct system of the biliary tract showed substantial similarity, reflected in the alpha and beta diversity indices and relative abundances.