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Background: Esfahan Province is considered as one of the main focus of zoonotic cutaneous leishmaniasis (ZCL) in Iran. Recently, ZCL distribution is expanding through this province leading to report of new cases in non-endemic areas. In the current study epidemiological aspects of ZCL has been investigated in Naein County in Esfahan Prov­ince. Methods: Adult sand flies were collected from beginning to the end of their seasonal activity. Rodents were caught by Sherman live traps once a month for one year. To active case detection, a hundred households in each selected village were visited in November and December 2016. Nested-PCR was employed to detect Leishmania parasite in the vector, reservoir and human. Results: Totally 1562 sand flies including Phlebotomus sergenti, Phlebotomus papatasi, Sergentomyia sintoni and Sergentomyia mervinae were collected and identified. No Leishmania infection was detected in the collected sand flies. All of the 30 collected rodents were identified as Rhombomys opimus and of these 3.3% and 26.7% were in­fected by Leishmania major using microscopic and molecular technique respectively. Totally, 914 individuals were investigated and the ulcer and scar rate of ZCL calculated to be at 1.1 and 15.3per 1000 population, respectively. Molecular results confirmed L. major infection in human and reservoir samples. Conclusion: It is concluded that ZCL is established in the area in low endemicity, and it is extrapolated the disease will not be a serious increasing health problem in near future in this region.