We have designed a new genotyping scheme for molecular diagnosis of the different Leishmania species pathogenic to humans. This scheme is based on PCR amplified sequences from the gene for the spliced leader RNA (mini-exon). This target was selected because it is present as tandem repeats (100 to 200 copies) in the genus Leishmania and other kinetoplastida, but is absent from the mammalian hosts and the sandfly vectors. The exon is highly conserved, whereas the intron and non-transcribed spacer region vary in size and sequence among different species. Thus, it was possible to amplify DNA from both Old and New World pathogenic Leishmania complexes using a single pair of primers deriving from the conserved region of the mini-exon tandem repeat. Species identification was performed by digesting mini-exon PCR products with one or two different restriction enzymes. Restriction fragment length polymorphism (RFLP) generated species-specific patterns of bands visualized in agarose gels, which allowed to differentiate each species unequivocally.