A reverse transcription-PCR (RT-PCR) assay was designed, according to previously determined and newly derived genetic data, to target S genomic segments of 47 viruses, including 29 arthropod-borne human pathogens, of the family BUNYAVIRIDAE: The analytical sensitivity of the presented assay was evaluated through its application to RNAs extracted from quantitated dilutions of bunyaviruses of interest. Additionally, the assay's analytical specificity was determined through the evaluation of RNAs extracted from selected bunyaviruses and other representative arthropod-borne viruses isolated from a diverse group of host species and temporal and geographic origins. After RT-PCR amplification, DNAs amplified from bunyaviruses of interest were subjected to a novel multiplex sequencing method to confirm bunyavirus positivity and provide preliminary, species-level S segment identification. It is our goal that this assay will be used as a tool for identification and characterization of emergent arthropod-borne bunyavirus isolates of medical import as well as related viruses of the family Bunyaviridae that have not been associated with human illness.