Real-time (fluorogenic) PCR assays provide a rapid method of assessing the threat arthropod-borne diseases pose to military forces deployed to remote locations. During recent deployments to Operation Iraqi Freedom we evaluated the use of a real-time PCR assay as a means of detecting Leishmania parasites in Phlebotomine sand flies. To date, a total of 4,313 pools containing 41,244 sand flies have been tested using a Leishmania-generic assay. Over 13% (585/4,313) of the pools were positive, resulting in a minimum infection rate of 1.4% (585/41,244). We subsequently re-tested these positive pools using assays specific to the Leishmania species causing old world leishmaniasis, i.e., L. major, L. tropica and visceral-complex assays -- 2.6% (14/541), 1.6% (8/507) and 3.1% (13/415) of the samples were positive for each specific assay, respectively. Over 90% of the Leishmania-generic positive samples were not positive when assayed with the three specific assays. Possible explanations for these results include: i) the generic assay is approximately 10-fold more sensitive than the species-specific assays , ii) Leishmania species other than the human pathogens (e.g., L. tarentolae) may have been present in the sand flies, or iii) inherent problems with the species-specific assays resulted in false negative results. We have initiated a study to determine the species of Leishmania parasites in sand flies from Iraq using homology of the glucose-6-phosphate isomerase gene. To date, a total of 25 isolates have been assessed --14 (56%) were L. tarentolae, three (12%) were L. infantum, one (4%) was related to both L. major and L. tropica, and 7 (28%) were undetermined. The implications of these results will be discussed.