Blood-engorged mosquitoes were collected at poultry farms during an outbreak of highly pathogenic avian influenza in Central Thailand during October 2005. These mosquitoes tested positive for H5N1 virus by reverse transcription-polymerase chain reaction (RT-PCR). Results were confirmed by limited sequencing of the H5 and N1 segments. Infection and replication of this virus in the C6/36 mosquito cell line was confirmed by quantitative real-time PCR. However, transmission by mosquitoes was not evaluated, and further research is needed. Collecting and testing mosquitoes engorged with the blood of domestic or wild animals could be a valuable tool for veterinary and public health authorities who conduct surveillance for H5N1 virus spread.