Leishmanioses are vector-borne diseases caused by Leishmania spp., which are transmitted by phlebotomine sand flies (Diptera, Psychodidae). The recent reports in humans of Leishmania tarentolae, which is primarily found in cold-blooded animals, and Leishmania infantum in Sergentomyia minuta spurred us to develop an internal transcribed spacer 1-based duplex quantitative real-time PCR (dqPCR) assay for the detection and differentiation between these Leishmania spp. The specificity of dqPCR was assessed by processing DNA samples from Phlebotomus spp. (n=188) and Se. minuta (n=171) and from tissues (i.e., heart, liver, muscle, lungs, spleen, kidney, eggs) of Podarcis siculus (n=4) and Tarentola mauritanica (n=3). In the absence of naturally infected and/or co-infected lizards, DNA from cultured L. infantum and L. tarentolae were spiked into tissues of lizards and used as controls. The analytical sensitivity of the dqPCR, assessed using 10-fold serial dilutions of DNA from both Leishmania spp. and spiked DNA samples from lizards was 2.3 x 10-7 ng/2 µl for L. infantum and 2.1 x 10-7 ng/2 µl for L. tarentolae. With the spiked DNA samples, the dqPCR detected up to 2.6 x 10-6 ng/2 µl of L. infantum and up to 2.1 x 10-7 ng/2 µl of L. tarentolae. Of 359 phlebotomine sand flies tested, five (3.6%) and two (1.4%) Ph. perniciosus scored positive for L. infantum and L. tarentolae, respectively. Similarly, of 171 Se. minuta, 56 (32.7%) and six (3.5%) scored positive for L. tarentolae and L. infantum, respectively. Co-infection with both Leishmania spp. was detected in two Se. minuta (1.2%). Out of seven reptiles tested, four P. siculus were positive for L. tarentolae. The newly dqPCR herein described may represent an improvement in the diagnosis of L. infantum and L. tarentolae and may assist in identifying the role of lizards as reservoirs and Se. minuta as vector, for these Leishmania spp.