Background: Leishmaniasis is a parasitic infectious disease with a wide spectrum in tropical and subtropical areas. Antimoniates are compounds of choice to treat cutaneous leishmaniasis in Iran and many other countries of the world. There are many fluctuations in response to leishmanicidal agents according to type of the disease, involved species of the parasite, and drug regimen. Methods: This study was conducted in the Skin Disease and Leishmaniasis Research Center, Isfahan, Iran. Novy-MacNeal-Nicolle (NNN) and Roswell Park Memorial Institute (RPMI) 1640 media were used to isolate parasites from skin lesions of patients. Deoxyribonucleic acid (DNA) extraction of Leishmania isolates was performed and strain identification was determined using specific internal transcribed spacers-1 (ITS-1) primers. After amplification, the resulted products were analyzed using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP). Patients were under traditional glucantime therapy and were followed up for 14 weeks to detect their treatment courses. Findings: PCR-RELP showed that all etiologic agents of cutaneous leishmaniasis (CL) among referral patients in Isfahan were L. major
