Background and Aim: Viral hepatitis is a health threat for hemodialysis (HD) patients and it may be transmitted during treatment. Some patients categorized to have viral hepatitis were found to be non-viremic. To clarify the discrepancy between the serological tests in HD patients, we conducted the study. Methods: A total of 1681 HD patients was included. Blood samples were analyzed for hepatitis B surface antigen (HBsAg) and anti-hepatitis C antibody (anti-HCV). Detection of hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA were performed in either HBsAg (+) or anti-HCV (+) samples. HBV DNA/HCV RNA was also measured in a subset of HBsAg (-) and anti-HCV (-) patients. Liver function tests were analyzed and compared with the serological and virological tests. Results: The serological tests showed that 230 patients (13.7%) were HBsAg (+) and 290 (17.3%) were anti-HCV (+). We were unable to detect HBV DNA in 97 of 230 (42.2%) HBsAg (+) patients, and HCV RNA could not be found in 76 of 290 (26.2%) anti-HCV (+) patients. In 167 HBsAg (-) patients, only one showed a trace amount of HBV DNA. None of 151 anti-HCV (-) patients showed detectable HCV RNA. The prevalence rate of viral hepatitis remains high in Taiwanese HD patients: 13.7% for HBV and 17.3% for HCV. However, virological analysis showed 42.2% non-viremic rate for HBsAg and 26.2% non-viremic rate for anti-HCV. Conclusions: The findings might challenge the presently suggested principles of bed and machine dedication and the diagnosis of viral hepatitis in HD patients.