Background: About 90% of cutaneous leishmaniasis (CL) cases are reported from 7 countries including Iran. In this study, the cutaneous leishmaniasis species causing CL in Gonabad, Bardaskan, Kashmar cities in central Khorasan (Iran) were identified by kDNA-PCR. Methods:During the study, 93 suspected patients with CL, who were referred to the dermatology research center in these cities, were evaluated based on age, clinical forms, and place of residence. Direct microscopy was employed for parasitological diagnosis and PCR from skin ulcers performed using specific kDNA primers. Data were analyzed using the SPSS software.Results: Among 93 individuals with skin ulcers suspected to CL, the results of 81 direct smears were positive. PCR bands were observed in 84 examined samples, of which 68 (81%) samples were identified for Leishmania tropica and 16 (19%) samples for L. major. 12 patients were from Bardaskan with 10 cases of L. tropica, 23 patients from Gonabad with 20 cases of L. tropica, and 49 patients from Kashmar with 38 cases of L. tropica. Most of the lesions were located on hands (37%), the most clinical feature was papule (75%), and most of patients were 21 -30 years old. Conclusions:Previous epidemiologic studies have indicated that anthroponotic cutaneous leishmaniasis is the only dominant CL in the center and south of Khorasan. However, this study introduced new foci of zoonotic cutaneous leishmaniasis in these areas. Kashmar city was introduced as a new focus of zoonotic cutaneous leishmaniasis.