Since 1998, multiple strains of bluetongue virus (BTV), belonging to six different serotypes (types 1, 2, 4, 8, 9 and 16) have caused outbreaks of disease in Europe, causing one of the largest epizootics of bluetongue ever recorded, with the deaths of >1.8 million animals (mainly sheep). The persistence and continuing spread of BTV in Europe and elsewhere highlights the importance of sensitive and reliable diagnostic assay systems that can be used to rapidly identify infected animals, helping to combat spread of the virus and disease. BTV has a genome composed of 10 linear segments of dsRNA. We describe a real-time RT-PCR assay that targets the highly conserved genome segment 1 (encoding the viral polymerase--VP1) that can be used to detect all of the 24 serotypes, as well as geographic variants (different topotypes) within individual serotypes of BTV. After an initial evaluation using 132 BTV samples including representatives of all 24 BTV serotypes, this assay was used by the European Community Reference Laboratory (CRL) at IAH Pirbright to confirm the negative status of 2,255 animals imported to the UK from regions that were considered to be at risk during the 2006 outbreak of BTV-8 in Northern Europe. All of these animals were also negative by competition ELISA to detect BTV specific antibodies and none of them developed clinical signs of infection. These studies have demonstrated the value of the assay for the rapid screening of field samples.