Endoscopic ultrasound (EUS) and ultrasound (US) are the two primary imaging modalities used to guide pancreatic needle biopsy. This study aimed to compare the diagnostic performance and complications associated with EUS- and US-guided pancreatic biopsies.A total of 2517 consecutive patients who underwent 2583 cases of EUS- or US-guided pancreatic biopsy for focal pancreatic disease between January 2017 and December 2023 at seven university teaching hospitals were included. The endpoints evaluated were diagnostic inaccuracy, complications, and repeat biopsies for each method. The propensity score matching (PSM) method was used for the analysis.For the entire cohort, diagnostic inaccuracies were observed in 7.0% of cases in the EUS-guided biopsy group compared to 3.5% in the US-guided biopsy group, representing a statistically significant difference (p = 0.001). The rates of major (p = 1.000) and minor complications (p = 0.309) were similar between the two groups. The rate of repeat biopsies was significantly higher in the EUS-guided biopsy group than in the US-guided biopsy group (5.0% vs. 2.8%, p = 0.024). However, after balancing lesion characteristics using PSM, no significant differences were observed between the EUS- and US-guided biopsies in diagnostic inaccuracy (7.2% vs. 8.5%, p = 0.600), major complications (0.5% vs. 0%, p = 0.499), minor complications (1.7% vs. 0.2%, p = 0.069), or repeat biopsy rate (5.5% vs. 6.5%, p = 0.656). Subgroup analysis revealed that among patients with exophytic and backward growth pancreatic lesions, both diagnostic inaccuracy (7.1% vs. 33.3%, p = 0.001) and the repeat biopsy rate (10.7% vs. 27.8%, p = 0.029) were significantly lower in the EUS-guided biopsy group compared to the US-guided biopsy group.This study confirmed that both EUS-guided and US-guided pancreatic biopsies are safe and effective for patients with focal pancreatic disease. After PSM, diagnostic inaccuracy, repeat biopsy and complication rates were similar, but EUS-guided biopsy was preferred for lesions with exophytic and backward growth morphology.Copyright © 2025 The Author(s). Published by Wolters Kluwer Health, Inc.