Lambton Public Health says mosquitoes collected from a trap in Corunna on August 23 have tested positive for West Nile virus.
West Nile had been confirmed earlier this year in Lambton County after positive results from a mosquito pool in Wyoming, and a crow in Sarnia.
The virus is spread to humans through the bite of an infected mosquito.
No human cases have been reported in Lambton.
Less than 1% of people infected with the virus get seriously ill.