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<i>Phlebotomus papatasi</i>, an Old World sand fly species, is primarily responsible for the transmission of leishmaniasis, a highly infectious and potentially lethal disease. International travel, especially military rotations, between domestic locations and <i>P. papatasi</i>-prevalent regions in the Middle East poses an imminent threat to the public health of US citizens. Because of its small size and cryptic morphology, identification of <i>P. papatasi</i> is challenging and labor-intensive. Here, we developed a ribosomal DNA-polymerase chain reaction (PCR)-based diagnostic assay that is capable of detecting <i>P. papatasi</i> genomic DNA from mixed samples containing multiple sand flies native to the Americas. Serial dilution of <i>P. papatasi</i> samples demonstrated that this diagnostic assay could detect one <i>P. papatasi</i> from up to 255 non-target sand flies. Due to its simplicity, sensitivity and specificity, this rapid identification tool is suited for a long-term surveillance program to screen for the presence of <i>P. papatasi</i> in the continental United States and to reveal geographical regions potentially vulnerable to sand fly-borne diseases.