Abstract Background The leishmaniases comprise a spectrum of clinical manifestations caused by different species of Leishmania. Identification of species is important for diagnosis, treatment and follow-up management. However, there is no gold standard for species identification. High resolution melting analysis (HRM) offers a possibility to differentiate Leishmania species without the need for processing of the PCR-product. The amino acid permease 3 (aap3) gene is an exclusive target for trypanosomatids and is conserved among Leishmania spp., thus it can be a valuable target for an HRM assay for diagnosis of the leishmaniases. Results The HRM dissociation profiles of three amplicons targeting the aap3-coding region allowed the discrimination of L. (Leishmania) donovani, L. (L.) infantum, L. (L.) major, L. (L.) tropica, L. (L.) mexicana, L. (L.) amazonensis, L. (Viannia) braziliensis, L. (V.) guyanensis, L. (V.) lainsoni, L. (V.) naiffi and L. (V.) shawi using DNA from promastigote cultures. The protocol was validated with DNA samples from clinical infection in humans and a cat, naturally infected sand flies, and experimentally infected mice. Conclusions HRM analysis using the aap3 coding sequence as target is a relatively cheap, fast and robust strategy to detect and discriminate Leishmania species from all the endemic regions worldwide. The target and method proved to be useful in clinical, field and experimental samples, thus it could be used as a tool in diagnosis as well as ecological and epidemiological studies.