West Nile Virus lineage 1 (WNV lin1) emerged in North-Eastern Italy in 2008 and, since then, it has been detected in animals, humans and mosquitoes. Three years later, in the same area, a lineage 2 (lin2) strain of WNV was found in birds and vectors. On August the 21st, during the 2012 WNV entomological surveillance plan, a WNV lin2 strain was detected by RT-PCR in a pool of Culex pipiens mosquitoes captured in Veneto region. According to the alignment of the partial sequences of the NS5 and NS3 genes, no differences between this Italian lineage 2 strain and the Nea Santa-Greece-2010 WNV isolate (Gr-10) were observed. Similarly to the Gr-10 strain, the putative NS3 amino acid sequences of the Italian strain showed proline in position 249 instead of histidine (H249P). Although proline in position 249 has been suggested to increase the virulence of WNV strains, neither human nor veterinary cases associated to this strain have been reported in the region. A prompt mosquito disinfestation was organized to avoid the spread of this potential threatening virus. The simultaneous circulation of both WNV lineage 1 and 2 confirms North-Eastern Italy as a high risk area for WNV emergence and highlights the need for a continuous surveillance.