A quantitative real-time RT-PCR assay for the detection of Colorado Tick Fever (CTF) viral RNA in human clinical samples is presented. The sensitivity of this assay has been shown to be greater than that of the isolation of virus in Vero cells by standard plaque assay in a direct comparison. The specificity of the CTF quantitative real-time RT-PCR assay was determined by the exclusive detection of CTF viral RNAs when applied to a diverse panel of CTF viral isolates and reference strain agents known to circulate in areas of CTF virus transmission. Lastly, the quantitative real-time RT-PCR assay demonstrated exceptional sensitivity for the detection of CTF viral RNA in acute human serum. The quantitative real-time RT-PCR assay is efficient, sensitive and specific and as such is useful for the detection of CTF viral RNA in the diagnostic or research laboratory.