Endoscopic ultrasound-guided tissue acquisition is vital for diagnosing pancreatic and peridigestive tract lesions. A new three-prong asymmetry tip needle has been developed for this procedure. In this study, we retrospectively assessed the diagnostic ability, tissue collection volume, and procedural adverse events of the three-prong asymmetry tip needle for solid pancreatic, subepithelial, and other organ lesions. We analyzed the data of 58 consecutive patients who underwent endoscopic ultrasound-guided tissue acquisition using a three-prong asymmetry tip needle between August 2022 and April 2023 at a single care center. The tissue collection rate was 91.4% with 89.7% accuracy, 89.3% sensitivity, 100% specificity, 100% positive predictive value, and 25% negative predictive value. No significant differences in collection rates or diagnostic performance were observed based on the target organ, puncture route, or lesion size. Using our original assessment method, the average histological core tissue score was 3.1 ± 0.8, whereas the blood contamination volume was 2.5 ± 0.8. Only one of 58 patients (1.7%) developed a pancreatic fistula of moderate severity as an adverse event. The three-prong asymmetry tip needle demonstrated good diagnostic capability and adequate sample volume with safety for pancreatic, subepithelial, and other organ lesions.