To gain insight into the strain(s) causing human cases of West Nile virus (WNV) infection in Greece in 2012, mosquitoes were collected and tested for probable WNV infection. WNV lineage 2 sequences were obtained from two of 14 Culex spp. pools collected in Xanthi Prefecture, and especially in the municipality with the highest incidence in Greece. As in the 2 previous years, the strain contains the H249P substitution in the NS3 protein. It seems that this specific strain is fully established in Greece and causes large-scale outbreaks. Thus, prevention and control measures have to be taken, while enhanced surveillance is needed.