Objective: The incidence of carcinoma of extrahepatic bile duct tends to increase during recent decade in China, but its cause is unclear. This study is to investigate the hepatitis C virus (HCV) and Hepatitis B virus (HBV) infection in the tissues of carcinoma of extrahepatic bile duct and study their correlation. Methods: HCV RNA and HBV DNA was detected by in situ polymerase chain reaction (IS-PCR) in sections of 51 cases of carcinoma of extrahepatic bile duct and 34 cases of control group. Results: Of 51 carcinoma of extrahepatic bile duct, HCV RNA was detected in 18 (35.4%), HBV DNA in 8 (15.9%). In 34 cases of control group, HCV RNA was detected in 2 (5.9%), and HBV DNA in 3 (8.8%). Conclusion: The prevalence of hepatitis C and B viral infection in the tissues of carcinoma of extrahepatic bile duct was significantly higher than in control group. The findings suggest a correlation between HCV, HBV infection and carcinoma of extrahepatic bile duct, inferring HCV and HBV might be involved in the development of carcinoma of extrahepatic bile duct.