The sequence of the genome segment 10 (Seg-10) encoding NS3/NS3A was determined for 19 field isolates of Bluetongue virus (BTV) of serotypes BTV-1, BTV-4, BTV-9 and BTV-16, derived from epizootics in Greece in the years 1979 and 1998-2001. The aim of the study was to define the molecular epidemiology of the virus in this part of the Mediterranean basin. On the basis of the Seg-10 sequences, the isolates grouped into two distinct phylogenetic clusters. These were Greek group I of solely serotype BTV-4 viruses, and Greek group II of serotypes BTV-1, BTV-9 and BTV-16 viruses. The isolates in Greek group I clustered with the Corsican and Tunisian BTV-2 serotypes and US group II strains of BTV-10 and BTV-13 serotypes, while those in Greek group II with Chinese, Indian and Australian viruses of different serotypes suggesting that viruses derived from two distinct ecosystems have caused BT incursions in Greece over the last 25 years. The NS3/NS3A sequences of most of the BTV-4 isolates were identical, irrespective of the year of isolation, geographical location and host species or tissue origin. Maximum of 15-16% nucleic acid sequence variation, but only 4% deduced amino acid substitution, were observed between groups I and II. Furthermore, the clustering of the NS3/NS3A sequences was independent of the viral serotype, indicating the occurrence of genome segment reassortment during the course of evolution of the viruses.