A LightCycler real-time polymerase chain reaction (PCR) assay was developed to detectWuchereria bancrofti DNA in blood-fed mosquitoes. The assay is based on fluorescence melting curve analysis of the PCR product generated from a family of repeated DNA elements: the 182 bp SspI repeat, specific to the genus Wuchereria. According to the melting temperature, W. bancrofti infected-mosquitoes were differentiated from Brugia malayi-infected and non-infected mosquitoes as well as from genomic DNA of Dirofilaria immitis and human DNA. The method proved to be 100% sensitive in all W. bancrofti-infected mosquitoes. Melting curve analysis offers a rapid alternative for the specific detection of W. bancrofti in mosquitoes. It is very accurate and sensitive, allows a high throughput and can be performed on very small samples. The method therefore has great potential for application in epidemiological studies.