In August 2000, bluetongue virus (BTV) appeared for the first time in Sardinia and, since then, the infection spread across Sicily and into the mainland of Italy involving at the beginning serotypes 2 and 9 and then, from 2002, 4 and 16. To reduce direct losses due to disease and indirect losses due to new serotype circulation, the 2004 Italian vaccination campaign included the modified-live vaccines against BTV-4 and 16 produced by Onderstepoort Biological Product (OBP), South Africa. Few months after the end of the campaign, BTV-16 was reported broadly in the country and the need of differentiating field from the BTV-16 vaccine isolate became crucial. In this study, the gene segments 2, 5, 6 and 10 of both the Italian and vaccine BTV-16 strains were sequenced and their molecular relationship determined. As sequences of segment 5 were those showing the highest differences (17.3%), it was possible to develop a new diagnostic tool able to distinguish the Italian BTV-16 NS1 gene from that of the homologous vaccine strain. The procedure based on the use of a RT-PCR and the subsequent sequencing of the amplified product showed a high degree of sensitivity and specificity when samples from either BTV-16 vaccinated or infected sheep were tested.