Lineage 2 West Nile virus (WNV) strains were reported for the first time in Europe in 2004. Despite an almost silent circulation around their entry point in Hungary, an upsurge of pathogenicity occurred in 2010 as 262 people suffered from neuroinvasive disease in Greece. This increase in virulence was imputed to the emergence of a His249Pro mutation in the viral NS3 helicase, as previously evidenced in American crows experimentally infected with the prototype lineage 1 North-American WNV strain. However, since 2003, WNV strains bearing the NS3Pro genotype are regularly isolated in Western-Mediterranean countries without being correlated to any virulent outbreak in vertebrates. We thus sought to evaluate the weight of the NS3 249Pro genotype as a virulence marker of WNV in an in vivo avian model of WNV infection. We therefore characterized three genetically-related Eastern-Europe lineage 2 WNV strains in day-old specific pathogen-free (SPF) chickens: Hun2004 and Aus2008 which are both characterized by a NS3 249His genotype, and Gr2011 which is characterized by a NS3 249Pro genotype. Unlike Hun2004 and Aus2008, Gr2011 was weakly virulent in SPF chicks as Gr2011-induced viremia was lower and waned quicklier than in the Hun2004 and Aus2008 groups. Overall, this study showed that the presence of a proline residue at position 249 of the viral NS3 helicase is neither sufficient nor necessary to confer pathogenicity to any given lineage 2 WNV strain in birds.