Leishmaniasis diagnosis in regions where multiple species exist should identify each species directly in the clinical sample without parasite culturing. The sensitivity of two PCR approaches which amplify part of the ssu rRNA gene and the ribosomal internal transcribed spacer (ITS), respectively, was determined using human and dog blood seeded with Leishmania promastigotes. ssu-rDNA-PCR was more sensitive than ITS1-PCR, however species identification was not possible by the former approach. When a nested ITS1-PCR was used its sensitivity equaled the ssu-rDNA-PCR. Digestion of ITS1 amplicon with the restriction enzyme HaeIII distinguished all medically relevant Leishmania species. ITS1-PCR was used to diagnose 162 local and imported suspected cases of leishmaniasis in Israel, the Palestinian Authority and Germany. 113 cases (69.7%) were positive by PCR and species identification was possible in 110 samples. Leishmania DNA was also amplified and identified at the species level from archived non-stained and Giemsa stained microscope slides.