We have developed a PCR assay that in a single reaction distinguishes between Leishmania infantum and Leishmania donovani strains on the basis of different size of the amplicon. The targeted intergenic region between putative biopterin transporter and nucleotide binding protein on chromosome 35 is highly variable, species-specific and can be amplified from clinical samples. Based on the assay, five tested Leishmania archibaldi and L. infantum strains from the Sudan and Ethiopia clearly belong to L. donovani, which is in accordance with a recent multifactorial analysis of these strains. The nucleotide sequence reported in this paper has been submitted to the GenBankTM with accession number EU068004.