West Nile virus (WNV) genomic RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) in six out of 57 mosquito pools collected in Southern Portugal, during the summer of 2004, yielding an infection rate (IR) of 2.8/1,000 mosquitoes. Phylogenetic analysis of a 217-nucleotide fragment of the NS5 coding region, amplified from Culex pipiens s.l. and Culex univittatus unfed females, demonstrated a close relationship with WNV strains circulating in the Mediterranean basin (Italy, 1998; France, 2000; Morocco, 2003). The data in this short report demonstrate the presence of WNV in mosquitoes in Southern Portugal and the need of permanent surveillance for viral activity within the country.