Cutaneous leishmaniosis (CL) is a major public health challenge in Iran. Overall, 18 provinces out of 31 of the country's provinces are in the endemic areas. The objective of the present study was to determine the presence of CL species in Sabzevar and Neghaab cities, Khorasan Razavi province, northeastern Iran. Overall, 280 samples were taken from suspected individuals referred to Sabzevar's and Neghaab's health centers during 2014-2016. Smear preparation, case detection and further identification by partial amplification of 7SL RNA and high-resolution melting curve analysis were done for all samples, followed by randomly polymerase chain reaction (PCR)-sequencing confirmation. Based on findings, the most infection rate was found in males with the age groups of 20-30 in both districts. All samples were characterized as Leishmania major, except one isolate in Neghaab closely related to Leishmania tropica. Various risk factors play roles in creating new foci of zoonotic CL (ZCL) caused by L. major in Sabzevar and Neghaab in northeastern Iran. Reports of the prevalence of CL in new foci make serious concerns about the incidence of cases and expansion of disease to the neighboring areas. Further measures are essential to control the disease in the regions.