We report here the first blood donation positive for West Nile virus (WNV) by nucleic acid amplification testing collected in north-eastern Italy in July 2012.Partial sequencing of the WNV RNA demonstrated identity with a WNV lineage 1a genome identified in the same area in 2011 and divergence from the strain responsible for the outbreak in northern Italy in 2008–09. These data indicate that WNV activity in northern Italy is occurring earlier than expected and that different WNV strains are circulating.