West Nile virus (WNV) is a flavivirus that causes neurological disorders in less than 1 % of infected subjects. Human cases of WNV-associated fever and/or neurological disorders have been reported in Italy since 2008. The first outbreak occurred in the northeastern region of Italy surrounding the Po River and was caused by the Po River lineage 1 strain, and since then, WNV infections have been reported in several regions of central Italy. Although the virus is highly genetically conserved, stochastic mutations in its genome may lead to the emergence of new strains, as was observed in Italy in 2011 with the identification of two new lineage 1 strains, the WNV Piave and WNV Livenza strains. To help further define WNV epidemiology in Italy, we describe a case of an Italian man living in the Po River area who developed fatal encephalitis in 2009 due to infection with the WNV Piave strain. This finding supports the notion that the Piave strain has been circulating in this area of Italy for 2 years longer than was previously believed.