Arboviruses in the genus Flavivirus are major causes of human disease worldwide, and yet they are widely under reported. This is partially due to the classical methods of detection, using virologic or immunologic techniques, which typically lack sensitivity and specificity or require biological containment facilities in order to manipulate infectious cultures. Molecular detection assays based on broadly reactive (degenerate) primers also tend to have differential and lower sensitivity for many flaviviruses. In this study, an assay was designed to test for many flaviviruses using species-specific and group-specific primers in a single reaction. This real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay combines flavivirus species-specific and group-specific TaqMan primers and probes (some previously described) into a single tube and subjected it to a standardized thermocycling conditions. The multiplex assay contains specific detection for St. Louis encephalitis virus, West Nile virus, and consensus sequences for tick-borne encephalitis complex groups: Russian Spring-Summer encephalitis and Central European encephalitis viruses. The assay also contained group-specific primers for Dengue I, II, III and IV viruses. Viral RNA was extracted from infected cell-culture derived stock viruses, field samples, or was synthesized as subgenomic target RNA molecules. All flavivirus species and sample types were detected by the multiplex assay. A sensitivity analysis of the assay suggested that the multiplex was no less able to detect low virus titer samples than the single-virus assay. This technique allows for a collection of specific assays to be used to screen for the presence of many flaviviruses of interest while saving labor and reagents, and without sacrificing sensitivity. The results demonstrated that these viruses can be screened for specifically in multiplex reactions using the rapid and sensitive method of real time qRT-PCR.