Endoscopic ultrasound-guided fine needle aspiration (EUS-guided FNA) is becoming a preferred modality for diagnosing and staging GI and mediastinal malignancies. Although experts advocate on-site cytopathology assessment for tissue sample adequacy, there are few data to support this claim. Our goal was to determine whether on-site cytopathology interpretation improves the diagnostic yield of EUS-guided FNA. EUS-guided FNA results from two university hospital centers were reviewed and compared. At center 1, where EUS-guided FNA was performed with a cytopathologist on site, the results of 108 consecutive patients were evaluated. At center 2, where a cytopathologist is unavailable, the results of 87 consecutive patients were reviewed. One endoscopist performed all procedures at both institutions. Cytologic diagnoses were categorized as positive or negative for malignancy, suspicious for malignancy, atypical/indeterminate, or unsatisfactory. The number of repeat procedures, needle passes, medication use, target site, age, and sex were compared between the two sites. Patients at center 2 were older (p = 0.04) and predominantly female (p = 0.03). Pancreas was the most common target site at center 2, whereas thoraco-abdominal nodes were the most common at center 1 (p = 0.0001). Patients at center 1 had a diagnosis of positive or negative for malignancy more frequently (p = 0.001) and were less likely to have an unsatisfactory specimen (p = 0.035) or repeat procedure, although the latter was not significant (p = 0.156). On-site cytopathology interpretation improves the diagnostic yield of EUS-guided FNA. EUS centers should allocate resources to cover for on-site cytopathology evaluation.