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Objective Acquiring adequate tissue for immunohistochemical (IHC) analysis is important in the differential diagnosis of subepithelial tumors (SETs). In this study, we aimed to compare the diagnostic yield based on IHC analysis between endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) and EUS-guided fine needle biopsy (EUS-FNB) after on-site cytological evaluation for cellularity in gastric SETs. Methods In 22 patients with gastric SETs, EUS-guided tissue sampling was performed on the same SET in a randomized sequence, with EUS-FNA followed by EUS-FNB, or vice versa. After on-site cytological evaluation for cellularity, the cytological and histological examinations were performed. Results There was a significant difference in the median number of needle passes to obtain adequate cellularity in the on-site cytological evaluation (2.0 for EUS-FNA vs 1.0 for EUS-FNB, P = 0.008). The proportion of adequate cellularity on the first needle pass was significantly higher in the EUS-FNB than in the EUS-FNA group (68.2% vs 31.8%, P = 0.034). However, diagnosis based on IHC examination was established in 15 (68.2%) and 18 (81.8%) cases by EUS-FNA and EUS-FNB, respectively ( P = 0.488). Conclusions EUS-FNB decreases the number of needle passes to obtain adequate cellularity and yields a higher proportion of adequate cellularity during the first needle pass compared with EUS-FNA in gastric SETs. However, there was no significant difference in diagnostic yield with IHC stain between the two procedures after on-site cytological evaluation for adequate cellularity. [ABSTRACT FROM AUTHOR]