BACKGROUND: Pancreatic neuroendocrine tumors (PanNETs) exhibit heterogenous behavior, whereby some small tumors are aggressive with a propensity for metastasis. Detection of somatic mutations associated with aggressive biology may help with patient stratification and surgical decision-making in patients with well-differentiated PanNETs. Using next-generation sequencing (NGS), we investigated the feasibility of detecting somatic mutations in endoscopic ultrasound-guided, fine-needle aspiration (EUS-FNA) specimens and determining the mutational concordance between the EUS-FNA specimens and the primary tumors. METHODS: Thirty-eight patients with well-differentiated, nonfunctioning PanNETs were obtained from two tertiary referral centers. Patient demographic characteristics and tumor, clinicopathologic features were collected. Tissue from both the EUS-FNA specimen and the primary tumor was extracted from archival tissue blocks. NGS using a panel of ten genes was performed on both samples. RESULTS: In our series, the median age was 61.1 years. Tumors were predominantly left-sided (60.5%) and unifocal (94.7%). The median tumor size was 2.2 cm. NGS detected somatic mutations in 29% of primary tumors and 36.8% of EUS-FNA specimens. In primary tumors, DAXX/ATRX mutations were predominantly detected (63.6%). In EUS-FNA specimens, MEN1 mutations were predominantly detected (64.3%). Among non-wild-type specimens, mutational concordance was achieved in 31.6% of cases. In 11 patients with a detectable mutation in the primary tumor, a mutation was detected in the EUS-FNA specimen in 45.5% of cases, with a mutational concordance of 54.5%. CONCLUSIONS: NGS can detect somatic mutations in EUS-FNA specimens of well-differentiated PanNETs. Efforts to improve detection sensitivity and mutational concordance are required to overcome current technical limitations.