Background and aim: Cutaneous leishmaniasis (CL) is a health problem of many countries in tropical and subtropical regions including Iran. Isfahan province is one of the foci of CL with the highest prevalence in Iran. This study was done to identify the species of Leishmania isolated from the patients in Ghohab Mohammad Abad, located in southwest of Isfahan which is a new foci of CL. Identification of the causative agent of CL is required to plan control measures and therapeutic strategies.Materials and methods: All residents of the village (18,477 individuals) were visited and interviewed to recruit patients with active lesion of CL. Direct samples and culture were taken from the suspected lesions for isolation and identification of Leishmania species. Identification was performed using kDNA minicircles in a PCR manner. Results: Forty three patients with suspected CL lesion were recruited and 25 parasitologically proven cases were identified. Eighteen isolates were used for identification and 7 isolates were excluded due to fungi contamination. All 18 isolates were characterized by PCR amplification to be Leishmania major.Conclusion: The etiologic agent of the CL in the region was identified to be L.major. Larger studies are needed to confirm that L. major is the rule etiologic agent of CL in this region.