Rotaviruses were detected by enzyme-linked immunosorbent assay (ELISA) in 92 out of 374 faecal samples collected between November 2003 and October 2004 at the Markaz Tebbi Koudakan Hospital, Tehran, Iran, from children aged 6 months to 5 years. Analysis of clinical and disease severity data showed a significant association between rotavirus infection and diarrhoea, vomiting and severe dehydration. Ninety-two samples (64 rotavirus ELISA-positive and 28 ELISA-negative samples) were sent to the Enteric Virus Unit, Virus Reference Department, Centre for Infection, Health Protection Agency, UK for rotavirus characterization by G-typing, P-typing and subgrouping (SG) using reverse transcriptase (RT)-PCR, semi-nested PCR and sequencing methods. In this study, both common and uncommon rotavirus genotypes were detected. The most prevalent types were G1P[8], SGII (59.2%) followed by G9P[8] SGII (15.5%) which has not been previously reported from Iran. Unusual genotypes G1P[10] SGI (2.8%) and G12P[8] SGII (1.4%) and strains derived from reassortment between common co-circulating genotypes such as G1P[4] SGII represented 5.6% of strains. Mixed infections with combinations of G1+G4P[8] SGII and G1+G9P[8] SGII were also found. This contrasts with previous reports from Iran in which a small number of common rotavirus strains (G1 and G4) were found. This study highlights the need for continued surveillance and characterization of rotaviruses to take account of the rapid evolution and introduction of novel rotaviruses into the human population.