This study described the first report of BTV-16 in Croatia. Serological evidence occurred in cattle at the end of September and continued during October and November 2004. All positive animals were in the Dubrovnik-Neretva County, a region located in the southernmost part of Croatia. BTV-16 infection was also detected in goats and sheep. Apart from few cases reported in Greece between 1999 and 2000, BTV-16 has never been reported in the Balkanic peninsula before. The BTV strain was isolated from cattle blood samples and typed as BTV-16. When the S5 was sequenced, it showed 100% homology with the BTV-16 vaccine isolate produced by Ondersterpoort Biological Product (SA) and used in Italy during the 2004 BT vaccination campaign. On the other hand no complete homology was found when the same RNA segment sequence was compared with that of the homologous Italian field isolate. As no evidence of livestock movements from Italy was demonstrated, an eolic transmission of the infection through infected Culicoides was hypothesised. According to the local meteostations, in several occasions, during the 2004 summer months, the west-east breeze blew with a speed above 50 km/h from Italy towards the Dubrovnik County. It is concluded that the BTV-16 which infected Croatian livestock was similar to the homologous OBP vaccine isolate and it is likely that it was introduced from Italy into the Southern regions of Croatia through infected Culicoides carried by the wind.