A naturally occurring mutation was detected within the probe binding region targeting the envelope gene sequence of West Nile virus used in real-time polymerase chain reaction assays to test mosquito pools and other samples. A single C→T transition 6nt from the 5′ end of the 16mer in the envelope gene probe-binding region at genomic position 1,194 reduced assay sensitivity. The mutation first was detected in 2009 and persisted at a low prevalence into 2011. The mutation caused a 0.4% false negative error rate during 2011. These data emphasized the importance of confirmational testing and redundancy in surveillance systems relying on highly specific nucleic acid detection platforms.