Routinely available clinical samples of all stages of pancreatic cancer could be used to elucidate its molecular mechanisms and identify novel therapeutic targets. We evaluated the use of next-generation sequencing (NGS) of endoscopically obtained pancreatic cancer tissues. We enrolled 147 patients who underwent endoscopic ultrasound-guided fine needle aspiration or endoscopic biopsy. The quantity of the extracted DNA was assessed and used for NGS of 78 cancer-related genes from which gene alterations and microsatellite instability (MSI) were extracted. NGS was successful in 141 out of 147 (96%) cases. Gene alterations were detected in 134 out of 141 (91%) samples, among which eight out of 10 samples with a DNA concentration below the detection limit had some type of gene alterations. Targetable genes were detected in 28 (19.9%) cases. MSI and germline mutations in homologous recombination repair associated genes were detected in 5% and 3% of cases, respectively. Cox regression analysis revealed that metastasis [P < 0.005; hazard ratio (HR), 3.30] was associated with poor prognosis in all pancreatic cancer patients and number of mutations <3 (P = 0.03; HR, 2.48) and serum carcinoembryonic antigen levels >5 ng/mL (P < 0.005; HR, 3.94) were associated with worse prognosis in cases without and with metastasis, respectively. Targeted sequencing of all stages of pancreatic cancer using available samples from real clinical practice could be used to determine the relationship between gene alterations and prognosis to help determine treatment choices.