West Nile virus (WNV) is a mosquito-borne flavivirus which causes important morbidity and mortality in birds, horses and humans. In the Western Mediterranean region, WNV causes sporadic, self-limited outbreaks, with few or no human cases. Here we report the characterization of two recent Western Mediterranean WNV isolates, obtained in Spain in 2007 from two golden eagles. Complete genome sequence comparisons revealed high identity between these isolates and close relationship with other Western Mediterranean WNV strains isolated since 1996. Phylogenetic analysis within this group indicated that two distinct phylogenetic groups have emerged from earlier strains. Pathogenicity analysis in mice showed that the Spanish isolate is less pathogenic than other strains either from the Western Mediterranean (Morocco 2003) or from North America (NY'99). Changes in amino acid position NS3-249 (claimed as a virulence marker) did not influence the pathogenicity observed.