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<i>Leishmania</i> protozoa are the etiological agents of visceral, cutaneous and mucocutaneous leishmaniasis. In specific geographical regions, such as Latin America, several <i>Leishmania</i> species are endemic and simultaneously present; therefore, a diagnostic method for species discrimination is warranted. In this attempt, many qPCR-based assays have been developed. Recently, we have shown that <i>L. (L.) infantum</i> and <i>L. (L.) amazonensis</i> can be distinguished through the comparison of the Cq values from two qPCR assays (qPCR-ML and qPCR-ama), designed to amplify kDNA minicircle subclasses more represented in <i>L. (L.) infantum</i> and <i>L. (L.) amazonensis</i>, respectively. This paper describes the application of this approach to <i>L. (L.) mexicana</i> and introduces a new qPCR-ITS1 assay followed by high-resolution melt (HRM) analysis to differentiate this species from <i>L. (L.) amazonensis</i>. We show that <i>L. (L.) mexicana</i> can be distinguished from <i>L. (L.) infantum</i> using the same approach we had previously validated <i>for L. (L.) amazonensis</i>. Moreover, it was also possible to reliably discriminate <i>L. (L.) mexicana</i> from <i>L. (L.) amazonensis</i> by using qPCR-ITS1 followed by an HRM analysis. Therefore, a diagnostic algorithm based on sequential qPCR assays coupled with HRM analysis was established to identify/differentiate <i>L. (L.) infantum</i>, <i>L. (L.) amazonensis</i>, <i>L. (L.) mexicana</i> and <i>Viannia</i> subgenus. These findings update and extend previous data published by our research group, providing an additional diagnostic tool in endemic areas with co-existing species.