ABSTRACTLeishmaniasis in humans is caused byLeishmaniaspp. in the subgeneraLeishmaniaandViannia. Species identification often has clinical relevance. Until recently, our laboratory relied on conventional PCR amplification of the internal transcribed spacer 2 (ITS2) region (ITS2-PCR) followed by sequencing analysis of the PCR product to differentiateLeishmaniaspp. Here we describe a novel real-time quantitative PCR (qPCR) approach based on the SYBR green technology (LSG-qPCR), which uses genus-specific primers that target the ITS1 region and amplify DNA from at least 10Leishmaniaspp., followed by analysis of the melting temperature (Tm) of the amplicons on qPCR platforms (the Mx3000P qPCR system [Stratagene-Agilent] and the 7500 real-time PCR system [ABI Life Technologies]). We initially evaluated the assay by testing referenceLeishmaniaisolates and comparing the results with those from the conventional ITS2-PCR approach. Then we compared the results from the real-time and conventional molecular approaches for clinical specimens from 1,051 patients submitted to the reference laboratory of the Centers for Disease Control and Prevention forLeishmaniadiagnostic testing. Specimens from 477 patients tested positive forLeishmaniaspp. with the LSG-qPCR assay, specimens from 465 of these 477 patients also tested positive with the conventional ITS2-PCR approach, and specimens from 10 of these 465 patients had positive results because of retesting prompted by LSG-qPCR positivity. On the basis of theTmvalues of the LSG-qPCR amplicons from reference and clinical specimens, we were able to differentiate four groups ofLeishmaniaparasites: theVianniasubgenus in aggregate; theLeishmania(Leishmania)donovanicomplex in aggregate; the speciesL. (L.)tropica; and the speciesL. (L.)mexicana,L. (L.)amazonensis,L. (L.)major, andL. (L.)aethiopicain aggregate.