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Background and study aims There are numerous studies published on the diagnostic yield of the new fine-needle biopsy (FNB) needles in pancreas masses. However, there are limited studies in suspected gastrointestinal stromal tumors (GIST lesions). The aim of this study was to evaluate the diagnostic yield of a new fork-tip FNB needle. Patients and methods This was a multicenter retrospective study of consecutive patients from prospectively maintained databases comparing endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) versus endoscopic ultrasound-guided FNB (EUS-FNB) using the fork-tip needle. Outcomes measured were cytopathology yield (ability to obtain tissue for analysis of cytology), ability to analyze the tissue for immunohistochemistry (IHC yield), and diagnostic yield (ability to provide a definitive diagnosis). Results A total of 147 patients were included in the study of which 101 underwent EUS-FNB and 46 patients underwent EUS-FNA. Median lesion size in each group was similar (21 mm vs 25 mm, P = 0.25). Cytopathology yield, IHC yield, and diagnostic yield were 92 % vs 46 % (P = 0.001), 89 % vs 41 % (P = 0.001), and 89 % vs 37 % (P = 0.001) between the FNB and FNA groups, respectively. Median number of passes was the same between the two groups at 3.5. Conclusion EUS-FNB is superior to EUS-FNA for diagnostic yield of suspected GIST lesions. This should be confirmed with a prospective study.