Pancreatic cancer is a lethal cancer with an increasing incidence. Despite improvements in chemotherapy, patients with pancreatic cancer continue to face poor prognoses. Endoscopic ultrasound-guided tissue acquisition (EUS-TA) is the primary method for obtaining tissue samples of pancreatic cancer. Due to advancements in next-generation sequencing (NGS) technologies, multiple parallel sequencing can be applied to EUS-TA samples. Genomic biomarkers for therapeutic stratification in pancreatic cancer are still lacking, however, NGS can unveil potential predictive genomic biomarkers of treatment response. Thus, the importance of NGS using EUS-TA samples is becoming recognized. In this review, we discuss the recent advances in EUS-TA application for NGS of pancreatic cancer.