Cutaneous Leishmaniasis is one of the health problems in Mediterranean regions including Iran. Considering the increase in the incidence of disease in Kashan, this study was designed to identify the Leishmania species in cutaneous leishmaniasis by PCR using primers of kinetoplastic DNA for treatment and appropriate measure for controlling of the disease. 130 patients suspected for cutaneous leishmaniosis referred to two health care centers of Kashan from 2012 to 2013 were examined. The demographic information as well as signs of the disease were recorded. The diagnosis of infection was based on observation of amastigotes within the smear of, serosity of the wound, after dying by Giemsa. Then, Leishmania species was identified following the extraction of DNA from serosity, and PCR with variable region of the KDNA. Expected PCR products of L. major and L. tropica were 650 bp and 760 bp respectively. The data were analyzed by SPSS using X2 and Kappa (K). Overall of 130 specimens, 87(66.9%) cases and 98 (75.4 %) cases were positive for microscopy and PCR methods respectively.  In PCR assay 70 (71.4 %) and 26 (26.6%) of the samples were identified as L. tropica and L. major respectively, and 2 (2 %) cases were detected as mix. The majority of the L.tropica or L. major had 1 to 2 wounds, but 11.5 % of L. major positive cases had more than 7 wounds respectively. Based on the results of this study, both species of Leishmania were present in Kashan, Therefore, it was suggested that careful preventive measure be taken in rural and urban parts. In addition, KDNA-PCR assay using serosity for accurate and quick diagnosis of cutaneous leishmaniasis is recommended.