BACKGROUND Hepatitis delta virus is a unique human pathogen responsible for some 20 million infections globally. This virus is dependent on hepatitis B virus for transmission and propagation. Currently, at least three genotypes of hepatitis delta virus with different geographic distribution and clinical manifestations are described. METHODS In this study, hepatitis delta virus RNA of 35 patients' sera were analyzed by RT- semi-nested polymerase chain reaction. Based on genomic differences of hepatitis delta antigen coding region of hepatitis delta virus RNA among hepatitis delta virus RNA-positive sera, the polymerase chain reaction products were digested with restriction enzymes and studied by restriction fragment length polymorphism. RESULTS Out of 35 samples, 13 (38.46%) were positive for hepatitis delta virus RNA by RT- semi-nested polymerase chain reaction. All polymorphisms were shown to be genotype I. Out of 13 hepatitis delta virus RNA-positive (13/35), eight were HBeAg negative. CONCLUSION Our data indicated that hepatitis delta virus isolates in Tehran are exclusively genotype I.