Objective: Mucosal leishmaniasis (ML) is a rare destructive disease that mainly affects the mucous membranes of the mouth and nose. The etiologic agent(s) of ML are not well known in the Middle East. Study Design: Cytologic smears of ML from the mucosal lesions of 7 patients were prepared by scraping. In 2 patients with nasal lesions, exfoliative cytology was made by washing the nasal cavity. The smears were both air dried and fixed in alcohol and stained. Scrapings from the same smears were then tested for leishmanial DNA by nested PCR. Results: This study characterized 9 isolates of ML, with 7 cases identified as Leishmania major and 2 as Leishmania tropica. While 6 patients were found to be positive by the cytology technique, the nested PCR was positive for all of these samples. Conclusions: Presence of granuloma and multinucleated giant cells in the negative smears of the patients who showed clinical manifestation of ML was an important clue for diagnosis of this disease. The PCR-based method not only appears to be a precise diagnostic approach in the identification of suspected cases of ML but is also efficient in determining the species of the parasite. L. major and L. tropica can lead to ML, but they result in different cytologic features.