Diagnosis of lesions of the gastrointestinal tract and adjacent structures is possible using an imaging modality, endoscopic ultrasonography (EUS). Fine-needle aspiration (FNA) is a suitable and cost-effective technique for obtaining cytohistologic material to confirm the diagnosis. EUS is capable of both characterizing the lesion and then guiding the FNA under real-time (RT) ultrasound guidance using a through-the-scope needle aspiration system. The goal of this study was to determine the diagnostic accuracy of this technique and to describe the clinicopathologic features. Sixty patients underwent EUS-guided RTFNA of 64 lesions, including pancreas (n = 45), periluminal lymph nodes (n = 12), mediastinal and retroperitoneal masses (n = 4), and hepatobiliary masses (n = 3). Follow-up data were obtained by surgery, histopathology, or clinical course. Thirty-one lesions were malignant, eight were atypical/suspicious, 16 were non-neoplastic, and nine were non-diagnostic. Of the 55 lesions with sufficient material for interpretation, 54 had follow-up confirmation of the RTFNA diagnosis. The calculated sensitivity and specificity for malignancy was 90% and 100%, respectively. Diagnostic accuracy for malignancy was excellent for gastrointestinal associated lymph nodes (100%), mediastinal and retroperitoneal masses (100%), somewhat less so for pancreatic tumors (94%), and poor for hepatobiliary lesions (33%). EUS-guided RTFNA is accurate for sampling small gastrointestinal tract-associated lesions. EUS-guided RTFNA should be considered as a procedure of choice in selected patients when the results will influence management decisions.