The aim of the study was to assess the 7SL RNA PCR followed by restriction fragment length polymorphism (RFLP) and reverse dot blot (RDB) assays for use in identification of Old World Leishmania species. Species-specific RFLP patterns were obtained for Leishmania major, Leishmania tropica and the Leishmania donovani complex when the 7SL RNA PCR product was digested with the restriction enzyme BsuRI, an isoschizomer of HaeIII. For the RDB assay, biotin-labelled 7SL RNA amplicons were hybridized to Leishmania genus-specific and species-specific oligonucleotide probes immobilized onto a membrane. The Old World Leishmania species could be distinguished by using five probes: one that was a genus-specific probe and hybridized to all Leishmania species (Lc), two that were specific for L. major (Lm1 and Lm2), one that was specific for L. tropica (Lt) and one that detected both L. major and L. tropica (Lmt). The PCR-RDB was 10 times more sensitive than 7SL PCR and can detect <1 parasite. In addition, the identification of species was easier and more reliable than with 7SL PCR-RFLP. 7SL PCR-RFLP detected parasites in 50 of 57 clinical samples, whereas PCR-RDB detected 53 and 55 were detected by amplification of kinetoplast (k) DNA. The 7SL RNA PCR has proven useful for direct diagnosis of Old World leishmaniasis, especially when combined with the RBD assay for species identification.