Background: Cytological diagnosis of pancreatic specimens obtained by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) is often challenging because of the small amount of sample or well-differentiated adenocarcinoma with weak cytological atypia. Therefore, the sensitivity and specificity of cytological diagnosis for pancreatic cancer should be improved. Hence, we aimed to evaluate the indices, which are used to distinguish malignant from benign lesions for the cytological diagnosis of pancreatic EUS-FNA specimens. Methods: Seven reviewers, including 3 cytotechnologists and 4 medical doctors, evaluated 20 morphological indices in pancreatic specimens obtained by EUS-FNA (malignant, n=111; benign, n=31). Statistical analyses were performed using Fisher’s exact test, logistic regression analysis, area under the receiver operating characteristic curve, and Youden Index.Results: Among the 20 indices, there was a high incidence rate (>40%) of the following 13 indices in malignant cases: structural atypia, hyperchromatic nucleus, irregular cell polarity, unclear cell boundary, nuclear membrane thickening, anisonucleosis, overlapping, irregular nuclei, high nuclear/cytoplasmic ratio, binding decline, simultaneous appearance of malignant and benign cells, enlarged nucleoli, and background necrosis. When we diagnosed pancreatic specimens using these 13 cytological indices, the cutoff value of 8/9 showed the highest Youden index (0.950) as well as high sensitivity and specificity in distinguishing malignant from benign specimens (98% and 97%, respectively). Conclusion: Thirteen cytological indices showed high sensitivity and specificity in differentiating malignant and benign lesions using pancreatic EUS-FNA samples. Further validation or prospective studies are necessary to establish criteria for the cytological diagnosis of pancreatic cancer.