Neonatal hepatitis is an inflammation of the liver that occurs after birth. Different risk factors including metabolic disorders, microbial infections, genetic disorders, and also idiopathic agents, may have role in neonatal hepatitis syndrome. Viruses which causing inflammation of the neonatal liver either before birth, through their mother, or shortly after birth, where detect in the most of infants who develop neonatal hepatitis. In this study the molecular and antigenic prevalence of some viral agents where investigated for determination of the possible role of viral infections in clinical complications of infants with neonatal hepatitis. In this retrospective study 26 paraffin embedded biopsy and autopsy tissues of 22 infants with neonatal hepatitis between years: 1996-2007 were collected. The genome prevalence of hepatitis type B and type C viruses (HBV and HCV), and human cytomegalovirus (HCMV) were analyzed by qualitative PCR protocols. Also the antigen presentation of HBV and HCMV were studied by immunohistochemistry (IHC) methods. The genome of rotavirus, HCMV and HCV were detected separately in 1 of 26(3.8 %) paraffin embedded autopsy and biopsy tissues. Also 3 of 26 (11.5 %) of this samples were infected with HBV genome. Antigens of HBV were diagnosed in 1 of 26 (3.8 %) neonatal samples by IHC protocols, but HCMV antigens were not detected in the samples of infants with neonatal hepatitis. Also co-infections of these viruses with together were detected in some samples of these neonates. Detection of separate and co-infections of HCMV, HBV, and HCV genomes in autopsy and biopsy tissues of infants with neonatal hepatitis and also detection of HBV antigens in these patients, announced the need of completed study for determination of accurate role of these viral infections in clinical outcomes of patients with neonatal hepatitis.