Reverse line blot hybridization assays (RLB) have been used for the rapid diagnosis and genotyping of many pathogens. The leishmaniases are caused by a large number of species, and rapid, accurate parasite characterization is important in deciding on appropriate therapy. Fourteen oligonucleotide probes, 2 genus specific and 12 species specific (2 specific for Leishmania major, 3 for L. tropica, 1 for L. infantum, 3 for L. donovani, and 3 for L. aethiopica), were prepared by using DNA sequences in the internal transcribed spacer 1 (ITS1) region of the rRNA genes. Probe specificity was evaluated by amplifying DNA from 21 reference strains using biotinylated ITS1 PCR primers and the RLB. The genus-specific probes, PP and PP3′, recognized all Leishmania species examined, while the species-specific probes were able to distinguish between all the Old World Leishmania species. Titrations using purified parasite DNA showed that the RLB is 10- to 100-fold more sensitive than ITS1 PCR and can detect <0.1 pg DNA. The RLB was compared to kinetoplast DNA (kDNA) and ITS1 PCR by using 67 samples from suspected cutaneous leishmaniasis (CL) patients in Israel and the West Bank. The RLB accurately identified 58/59 confirmed positive samples as CL, a result similar to that found by kDNA PCR (59/59) and better than that by ITS1 PCR (50/59). The positive predictive value and negative predictive value of the RLB were 95.1% and 83.3%, respectively. L. major or L. tropica was identified by the RLB in 55 of the confirmed positive cases, a level of accuracy better than that of ITS1 PCR with restriction fragment length polymorphism (42/59). Thus, RLB can be used to diagnose and characterize Old World CL.