The complete nucleotide sequences of the VP2 segments of bluetongue virus (BTV) isolates recovered from Italy, Greece and Israel, from 1998 to 2003, were determined. Phylogenetic analysis of these sequences, those from related viruses and the South African vaccine strains, were used to determine the probable geographic origin of BTV incursions into Italy. Results indicated that viruses from each of the four serotypes isolated in Italy (2, 4, 9 and 16) possibly had a different origin. Analysis of the bluetongue virus serotype 2 (BTV-2) isolates gave evidence that this serotype probably moved from Tunisia. BTV-4 results showed probable incursion from the southwest and not from Greece or Israel. BTV-9 isolates clearly have an eastern origin (most probably Greece), whereas BTV-16 isolates are indistinguishable from the BTV-16 live attenuated vaccine strain. The phylogenetic findings were supported by polyacrylamide gel electrophoresis (PAGE) analysis of the complete amplified genome of each isolate except for BTV-16 Italian field isolate, which showed a slightly different PAGE profile. A combination of the complete VP2 sequencing and PAGE analysis of complete genomes, allowed not only phylogenetic analysis, but also vaccine detection and assessment of reassortment events.