Leishmaniasis is caused by parasitic protozoa of the genus Leishmania. Cutaneous leishmaniasis (CL) is a complex disease with wide spectrum of clinical manifestations. In order to identify leishmania species causing CL in Isfahan by a definite molecular technique (PCR method), this study was undertaken over 2010- 2011. 124 Patients with suspicious lesion of Leishmaniasis and positive direct smear from lesion were selected. Samples were cultured in NNN and RPMI 1640 media Negative and positive control and clinical samples was applied for PCR in the same condition. In the next step, standard PCR was carried out using classic protocol. From 124 patients, 111 (89.51%) cases were infected as L. major and 12 (9.67%) cases were infected by L. tropica, However only in one patient simultaneous infectious with both L. major and L. tropica was identified by PCR techniques which could not be possible in microscopy. L.major was the most prevalent species in the studied patients (p-value<0.001).