| Summary: | The parasite protozoan <i>Leishmania</i>, the causative agent of leishmaniasis, includes two subgenera of medical interest: <i>Leishmania</i> (<i>Leishmania</i>) and <i>Leishmania</i> (<i>Viannia</i>). Parasite species detection and characterization is crucial to choose treatment protocols and to monitor the disease evolution. Molecular approaches can speed up and simplify the diagnostic process. In particular, several molecular assays target the mitochondrial DNA minicircle network (kDNA) that characterizes the <i>Leishmania</i> genus. We previously proposed a qPCR assay targeting kDNA, followed by high resolution melt (HRM) analysis (qPCR-ML) to distinguish <i>L.</i> (<i>L</i>.) <i>infantum</i> and <i>L.</i> (<i>L</i>.) <i>amazonensis</i> from <i>L. Viannia</i> species. Successively, this assay has been integrated with other qPCR assays, to differentiate <i>L.</i> (<i>L</i>.) <i>infantum</i>, <i>L.</i> (<i>L</i>.) <i>amazonensis</i> and <i>L.</i> (<i>L</i>.) <i>mexicana.</i> In this work, we tested the applicability of our qPCR-ML assay on <i>L.</i> (<i>L</i>.) <i>donovani</i>, <i>L.</i> (<i>L</i>.) <i>major</i>, <i>L.</i> (<i>L</i>.) <i>tropica</i> and <i>L.</i> (<i>L</i>.) <i>aethiopica</i>, showing that the qPCR-ML assay can also amplify Old World species, different from <i>L.</i> (<i>L</i>.) <i>infantum</i>, with good quantification limits (1 × 10<sup>−4</sup>–1 × 10<sup>−6</sup> ng/pcr tube). Moreover, we evaluated 11 <i>L.</i> (<i>L</i>.) <i>infantum</i> strains/isolates, evidencing the variability of the kDNA minicircle target molecules among the strains/isolates of the same species, and pointing out the possibility of quantification using different strains as reference. Taken together, these data account for the consideration of qPCR-ML as a quantitative pan-<i>Leishmania</i> assay.
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