The 5′ nuclease PCR assay uses a fluorescently labeled oligonucleotide probe (TaqMan) to rapidly detect and quantitate DNA templates in clinical samples. We developed a 5′ nuclease PCR assay targeting the plasminogen activator gene (pla) ofYersinia pestis. The assay is species specific, with a detection threshold of 2.1 × 105 copies of thepla target or 1.6 pg of total cell DNA. The assay detectedY. pestis in experimentally infected Xenopsylla cheopis fleas and in experimentally infected monkey blood and oropharyngeal swabs. The TaqMan assay is simple to perform and rapid and shows promise as a future field-adaptable technique.