Background and study aims Recently, a new Franseen design endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) needle was developed with the goal of providing more tissue for histology. We compared the tissue adequacy rate and nucleic acid yield of 22G EUS-FNB vs. 22G endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA), in solid gastrointestinal and extra-intestinal lesions. Patients and methods We conducted a randomized crossover study and recruited 36 patients. We performed three passes for pancreatic lesions and two passes for other lesions, using each needle. We blinded the pathologist to needle assignment. We assessed the diagnostic tissue adequacy rate and compared the total tissue area, diagnostic tissue area, and desmoplastic stroma (DS) area in cases of carcinoma. We also examined the nucleic acid yield of the two needles in pancreatic lesions. Results The lesions included 20 pancreatic masses (55 %), six gastric subepithelial lesions (17 %), five lymph nodes (14 %) and five other abdominal masses (14 %). Mean ± SD lesion size was 3.8 ± 2.0 cm. The final diagnosis was malignant in 27 lesions (75 %) and benign in nine lesions (25 %). We found EUS-FNB procured significantly more median total tissue area (5.2 mm 2 vs. 1.9 mm 2 , P < 0.001), diagnostic tissue area (2.2 mm 2 vs. 0.9 mm 2 , P = 0.029), and DS area (2 mm 2 vs. 0.1 mm 2 , P = 0.001) in lesions diagnosed as carcinoma (n = 23), as compared to EUS-FNA. In pancreatic lesions, EUS-FNB obtained significantly more nucleic acid than EUS-FNA (median; 4,085 ng vs. 2912 ng, P = 0.02). There was no difference in the cellblock or rapid on-site cytological evaluation (ROSE) diagnostic yield between the needles. Conclusion The 22G EUS-FNB provides more histological core tissue and adequate nucleic acid yield compared to 22G EUS-FNA. In this study, the diagnostic performance was similar between the needles.