Objective: Although cytology testing is considered a valuable method to diagnose tumors that are difficult to access such as hepato-biliary-pancreatic (HBP) malignancies, its diagnostic accuracy remains unclear. We therefore aimed to investigate the diagnostic accuracy of cytology testing for HBP tumors. Study Design: We performed a retrospective study of all cytology samples that were used to confirm radiologically detected HBP tumors between 2002 and 2016. The cytology techniques used in our center included fine needle aspiration (FNA), brush cytology, and aspiration of bile. Sensitivity, specificity, positive and negative predictive values, and likelihood ratios were calculated in comparison to histological confirmation. Results: From a total of 133 medical records, we calculated an overall sensitivity of 76%, specificity of 74%, a negative likelihood ratio of 0.30, and a positive likelihood ratio of 2.9. Cytology was more accurate in diagnosing lesions of the liver (sensitivity 79%, specificity 57%) and biliary tree (sensitivity 100%, specificity 50%) compared to pancreatic (sensitivity 60%, specificity 83%) and gallbladder lesions (sensitivity 50%, specificity 85%). Cytology was more accurate in detecting primary cancers (sensitivity 77%, specificity 73%) when compared to metastatic cancers (sensitivity 73%, specificity 100%). FNA was the most frequently used cytological technique to diagnose HBP lesions (sensitivity 78.8%). Conclusion: Cytological testing is efficient in diagnosing HBP cancers, especially for hepatobiliary tumors. Given its relative simplicity, cost-effectiveness, and paucity of alternative diagnostic methods, cytology should still be considered as a first-line tool for diagnosing HBP malignancies.