The utility of endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) for gastric subepithelial lesions (SELs) has been reported. In this study, we examined the optimal number of needle punctures during EUS-FNB for gastric SELs without rapid on-site evaluation (ROSE). The factors that allowed for a single needle puncture to arrive at the correct diagnosis were also analyzed. We conducted a retrospective study of all patients who underwent EUS-FNB to evaluate gastric SELs between April 2015 and September 2020; 51 patients with 57 gastric SELs were enrolled. The optimal number of needle punctures was determined when additional needle passes did not increase diagnostic sensitivity by more than 10%. Factors allowing for only a single needle puncture to arrive at the correct diagnosis were identified by univariate and multivariate logistic regression analyses. EUS-FNB resulted in a definitive final diagnosis in 48 of 57 lesions (84%). Lesions in the gastric body (odds ratio [OR] 6.15, 95% confidence interval [CI] 1.75-21.6; P < 0.01) and lesions punctured using a 22G Franseen needle (OR 3.61, 95% CI 1.07-12.3; P = 0.04) were independent factors that allowed for only a single needle puncture to arrive at the correct diagnosis. The optimal number of needle punctures for lesions using a 22G Franseen needle in the gastric body and other lesions was two and three, respectively. The optimal number of needle punctures in EUS-FNB for gastric SELs without ROSE was two or three, depending on the location and type of needle used.