The discrimination of Leishmania species from patient samples has epidemiological and clinical relevance. In this study, different gene target PCR-restriction fragment length polymorphism (RFLP) protocols were evaluated for their robustness as Leishmania species discriminators in 61 patients with cutaneous leishmaniasis. We modified the hsp70-PCR-RFLP protocol and found it to be the most reliable protocol for species identification.H uman infections by Leishmania spp. produce a pleiomorphic syndrome in which symptomatology depends on the parasite species and the immunological stage of the host. The symptoms range from completely asymptomatic to cutaneous, mucocutaneous, and visceral (1). Several authors have reported differences in the treatment outcomes linked to the parasite species (2-5). Furthermore, mucocutaneous leishmaniasis (MCL) is a belated complication associated with specific parasite species (3, 6) most commonly occurring in infections caused by the Leishmania (Viannia) subgenus. American cutaneous leishmaniasis (ACL) cases are usually the result of infections produced by this subgenus, and species identification is useful for treatment and prognosis. Molecular techniques may become a routine way to confirm suspected cases of ACL (7-10); the present study describes the best PCR-restriction fragment length polymorphism (RFLP) gene target for determining the species of Leishmania present in clinical samples from ACL lesions in a set of Colombian patients.The study was approved by the boards of ethical conduct of the Hospital Militar Central-Bogota-Colombia (HOMIC) and Centro Dermatologico Federico Lleras Acosta Bogota-Colombia (CDFLL) in accordance with national (resolution 008430 of the Colombian Health Ministry) and international (Declaration of Helsinki and amendments, World Medical Association, South Korea, 2008) guidelines. DNA was extracted from skin biopsy specimens from the internal border of the lesions from 42 adult patients with a clinical diagnosis of ACL. The diagnosis was confirmed microscopically in 35 patients and by PCR detection of the parasite in 7 patients. All patients voluntarily participated in the study and signed an informed consent.The CDFLL biobank provided 19 Giemsa-stained slide smears from cutaneous lesions. In 17 of them, the presence of Leishmania sp. amastigotes was microscopically confirmed, and in 2 smears, the detection of the parasite was established by PCR. DNA was recovered from the Giemsa-stained smears.All PCRs performed included DNA from 2 negative-control patients from CDFLL (with confirmed diagnoses of sporotrichosis and ecthyma gangrenosum) and from three healthy volunteers. The entire group of patients had been infected within the Colombian borders.We selected genes and sequences previously reported to be useful markers for species identification by PCR-RFLP of Leishmania species for further evaluation. We analyzed zinc-metalloprotease (gp63) (11), spliced leader (SL) (12), cysteine protease B1 (cpb) (13), and heat shock protein 70 (hsp70) (14, 15) for the...