Intrahepatic cholangiocarcinomas (IHCs) are rare in the United States, but the prevalence is steadily increasing and risk factors are poorly understood. Tissues were obtained at the time of surgical resection, and 11 cases of IHC were retrospectively studied for the presence of hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA. Hemi-nested and real-time polymerase chain reaction assays were used to detect HBV DNA, and nested reverse transcriptase-polymerase chain reaction was used to detect HCV RNA. Genotypes were determined for both HBV and HCV. The cases were predominantly from women (10/11), with an average age at surgery of 63 years and an average tumor size of 6 cm. Three cases (27%) were positive for either HBV or HCV nucleic acids: HBV alone (n = 1), HCV alone (1), coinfection with HBV and HCV (1). Both HBV-positive cases were genotype A, and both HCV cases were genotype 1a. Hepatitis B and C viral copy numbers were low in all cases. Evidence for active HBV replication was found in both HBV-positive cases, as they were positive for covalently closed circular DNA. In this study, 27% of ICC cases contained HBV and/or HCV nucleic acids, suggesting an etiologic role for these viruses in some cases of IHC.