Bluetongue virus (BTV), a member of the Orbivirus genus, has contributed to great economical losses in countries across the Mediterranean basin. Although BTV has an African origin, it has been reported in the South of Europe since 1924 when it was first detected in Cyprus. After this first Bluetongue (BT) outbreak, many others followed in most Mediterranean countries, resulting in seven BTV serotypes detected in 17 countries in Central-, South-Europe and North Africa in the last 10 years. Currently, six BTV serotypes (1, 2, 4, 8, 9 and 16) are circulating throughout Central- and Western Europe. The unexpected occurrence of BTV serotype 8 in Central Europe in 2006 and its spread and persistence have evidenced changes in the BTV scenario: new serotypes, never detected in the Mediterranean area so far, are playing a central role in the maintenance of BTV in Europe, and new species of Culicoides are now confirmed to be able to transmit the virus. Therefore, it is necessary to improve and implement specific assays in order to identify the serotypes currently present in different Mediterranean countries in a fast and reliable way. In this study, we present a new gel-based and real-time RT-PCR assay for the detection of BTV serotype 4. The sequence amplified in this test is located within BTV segment 2, a variable region of BTV dsRNA genome encoding the major outer-capsid protein VP2. No cross-reaction has been shown with other genetically or geographically related viruses and the sensitivity of this test allows the detection of 1.5-15 TCID50/ml of BTV.