Cholangiocarcinoma (CCA) and gallbladder carcinoma (G.BC) are biliary tract cancers with poor five-year survival and high recurrence rates.Both CCA and GBC patients suffer from lack of circulating diagnostic biomarkers at the early stage.Extracellular vesicles,especially exosomes,have been emerged as promising diagnostic sources for cancers due to.easy and quick accessibility.Hence,identification of exosomal biomarkers provides a novel strategy for CCA and GBC diagnosis.Here,five CCA patients and four GBC patients were enrolled for exosomal small RNA sequencing.Our data showed that exosomal piwi-interacting RNA (piRNA) populations were altered in the plasma of CCA and GBC patients.In comparison to healthy individuals,694 and 323 piRNAs were upregulated in CCA and GBC,respectively,while 36 and 191 piRNAs were downregulated.Interestingly,sequencing results predicted that piR-2660989,piR-10506469,piR-20548188,piR-10822895,piR-hsa-23209,and piR-18044111 were upregulated in both CCA and GBC plasma.Importantly,we further included blood samples from 50 health individuals,40 CCA patients,and 25 GBC patients and found that piR-10506469 were significantly increased in the exosomes of plasma from both CCA and GBC patients.Moreover,we analyzed the expression levels of differentially expressed exosomal piRNAs in the plasma of CCA and GBC patient before and after surgeries and found that piR-10506469 and piR-20548188 were significantly decreased in patients underwent surgeries.Taken together,our data revealed that exosomal piRNAs those are differentially expressed in CCA and GBC plasma may serve as potential biomarkers for the diagnosis of CCA and GBC.