The National Rotavirus Reference Centre together with collaborating laboratories Australia-wide has conducted rotavirus surveillance since June 1999. This report describes the serotypes of rotavirus strains responsible for the hospitalisation of children with acute gastroenteritis during the period 1 July 2003 to 30 June 2004. We examined 688 faecal samples using monoclonal antibody immunoassays, reverse transcription-polymerase chain reaction and polyacrylamide gel analysis. This revealed that serotype G1 has re-emerged as the major serotype nationally, representing 40 per cent of all strains, followed by serotype G3 (25.7%) serotype G2 (17.1%) and serotype G9 (11.7%). However, there is substantial geographic variation in the prevalence of rotavirus serotypes. These findings have implications for vaccine development strategies which have targeted prevention of disease due to serotypes G1-G4.