Mosquito-borne flaviviruses are emerging pathogens of increasing global public health concern due to their rapid increase in geographical range, and the impact of climate change. Japanese Encephalitis virus (JEV) and West Nile virus (WNV) are of concern due to the risk of re-emergence and introduction by migratory birds. Human cases due to WNV have never been reported, and those of JEV are rare in Singapore. Four sentinel vector surveillance sites were established in Singapore to understand the potential risk posed by these viruses. The present study revealed active JEV transmission in Singapore through the detection of JEV genotype II inCulex tritaeniorhynchus collected from an avian sanctuary. ACulex flavivirus, similar to the Quang Binh virus isolated fromCulex tritaeniorhychus in Vietnam andCxFV-LSFlaviV-A20-09virus isolated in China, was also detected inCulex spp. (vishnui subgroup).No WNV was detected. This study demonstrates the important role that surveillance plays in public health, and confirms the circulation of JEV among wildlife in Singapore despite the absence of reported human cases. Surveillance, collaboration between public health and wildlife managers, and control of mosquito population remain the key measures in risk mitigation of JEV transmission in the enzootic cycle between birds and mosquitoes.