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Differentiation between benign and malignant portal vein thrombosis (PVT) in the setting of a hepatocellular carcinoma (HCC) is of paramount importance. Histological analysis is usually not carried out because of potential severe side effects of the percutaneous approach. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) may be safer and may guide the clinical management of patients with HCC.To describe the feasibility of the EUS-FNA in a series of patients with HCC and PVT.A chart review of patients with PVT was performed from 2014 to 2016 in three tertiary care hospitals of Spain. Patients with chronic liver disease and PVT with a suspicion of HCC referred for EUS-FNA were included. The impact of the EUS-FNA was assessed by comparing staging following the Barcelona Clinic Liver Cancer algorithm (BCLC) before and after EUS-FNA.Of 104 patients with PVT and chronic liver disease, 23 were considered candidates for EUS-FNA. Eight patients were referred for EUS-FNA. The technique was feasible in seven patients and FNA was positive in six patients. No side effects were reported. EUS-FNA upstaged six out of seven (85.7%) patients: one patient BCLC stage B, two patients BCLC stage A, and three patients in whom the HCC was not diagnosed before EUS-FNA of the PVT. A benign PVT was found in the explant of the only patient with a negative PVT.EUS-FNA is a valuable technique in selected patients with chronic liver disease with PVT. It is feasible, safe, and may alter the clinical management in these patients.