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Aim: Cutaneous leishmaniasis (CL) is one of the most important health problems that are capable of involving both tropical and subtropical areas, especially in Iran. This cross-sectional study aimed to differentiate the species that are able to cause CL in Zahedan city by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Materials and Methods: It was conducted on 145 suspected CL patients in Zahedan city between 2014 and 2016. The smears were initially prepared, air-dried, fixed with absolute methanol, and stained with 10% Giemsa. Then, we examined the stained samples by a light microscope under 1000× magnifications. PCR assay targeted cytochrome b (cyt b) gene using LCBF1 and LCBR2 primers and the products digested by Ssp1 enzymes. Results: From 145 suspected CL patients, 76 (52.4%) were positive in microscopic examination. In addition, we detected gene of interest (cyt b) in 98 (67.5%). The results of PCR-RFLP indicated that 53/98 (54%) cases were Leishmania major and 45/98 (46%) were Leishmania tropica, and the main species in these areas was L. major. Conclusion: We concluded that the microscopic examination is not sensitive enough and is not able to distinguish between different Leishmania species. Instead, molecular methods like PCR-RFLP can be appropriately used with promising results. [ABSTRACT FROM AUTHOR]