There is a complete lack of highly sensitive and specific biomarkers for early pancreatic ductal adenocarcinoma (PDAC) diagnosis, limiting multi-modal therapeutic options. Mitochondrial DNA (mtDNA) is an excellent resource for biomarker discovery because of its high copy number and increased mutational frequency in cancer cells. We examined if mtDNA mutations can be detected in circulating extracellular vesicles (EVs) of PDAC patients and used for discerning between cancer and non-cancer subjects. A greater yield of circulating EVs (~ 1.4 fold; p = 0.002) was obtained in PDAC patients (n = 20) than non-cancer (NC) individuals (n = 10). PDAC-EVs contained a higher quantity of total DNA (~ 5.5 folds; p = 0.0001) than NC-EVs and had greater enrichment of mtDNA (~ 14.02-fold; p = 0.0001). PDAC-EVs also had higher levels of cardiolipin (a mitochondrial inner-membrane phospholipid), suggestive of their mitochondrial origin. All mtDNA mutations in PDAC-EVs were unique and frequency was remarkably higher. Most mtDNA mutations (41.5%) in PDAC-EVs were in the respiratory complex-I (RCI) (ND1-ND6), followed by the RCIII gene (CYTB; 11.2%). Among the non-coding genes, D-Loop and RNR2 exhibited the most mutations (15.2% each). Altogether, our study establishes, for the first time, that mtDNA mutations can be detected in circulating EVs and potentially serve as a tool for reliable PDAC diagnosis.