© 2018 American Society for Gastrointestinal Endoscopy Background and Aims: Two second-generation, flexible EUS fine-needle biopsy (FNB) needles have been marketed recently in the United States. Thus far, there have been no comparative studies of the diagnostic yield of these needles. The aim of this study was to compare the diagnostic yield achieved with FNB by using 1 needle during 1 time period and the other needle during a second time period. Methods: Consecutive patients with solid lesions undergoing EUS-FNB by using 1 of two 22-gauge FNB needles (Franseen needle or fork-tip) at 2 different time intervals were included. The final diagnosis was based on positive pathology results. In cases of a negative pathology result, the final diagnosis was based on clinical and imaging follow-up. Results: A total of 194 lesions (97 in each group) were sampled in 179 patients. Rapid on-site evaluation (ROSE) was used in 12% of cases. The overall diagnostic yield was lower in the Franseen needle group compared with the fork-tip needle group (61/97 [63%] vs 75/97 [77%], odds ratio (OR) 2.01, 1.07-3.8; P =.027). Similarly, subanalysis of the yield for solid pancreatic masses demonstrated a lower yield with the Franseen needle (34/53 [64%] vs 40/47 [85%], OR 3.4, 9.1-8.9; P =.017). Multivariate analysis controlling for the number of passes, site, and lesion size did not have any effect on diagnostic yield. There were no adverse events in either group. Conclusion: In this first, large, single-center comparative cohort study of 2 new, second-generation EUS-FNB needles of different design, the diagnostic yield when used primarily without ROSE was high in both groups but was significantly higher when a fork-tip needle was used.