Cutaneous leishmaniasis in Iran is usually caused by Leishmania major or L. tropica . However, the direct examination or the cultures of biopsies for diagnosis are not very sensitive. The objective of this study was to identify the responsible species obtained from patients suspected of cutaneous leishmaniasis referred to the reference laboratory at Yazd in Iran during 2010-2011 using parasitological and molecular assays. After completing a clinical/epidemiologic data questionnaire for 145 patients with suspected skin lesions, scraping samples were collected. Each specimen was examined using both direct microscopy and molecular assay using polymerase chain reaction-restriction length polymorphism (PCR-RFLP). Location of the lesions included 47.7% on hands, 30.7% on face, 15.4% on feet, and the remainder on other regions. Out of 145 samples, Leishman body was observed in 52 by direct smear and 73 by PCR assay. Molecular assay indicated 36 cases as L. major , 36 cases as L. tropica and one case as unknown. In conclusion, molecular characterization showed changing profile of Leishmania species in the study area which may have implications on treatment and/or control strategies.