Background: Isfahan is one of the major foci of cutaneous leishmaniasis in Iran. According to the epidemiological studies, both urban and rural forms have been reported in Isfahan. Accurate identification and characterization of Leishmania is essential for the control of the disease. Several different molecular techniques for Leishmania identification are developed including use of monoclonal antibodies. Objective: In the present study specific monoclonal antibodies against L.major (T1, T9), L.tropica (T10, T11) and L.donovani (D2) were used to identify and characterize the leishmania parasites isolated from patients with cutaneous leishmaniasis in Isfahan. Patients and Methods: Samples were obtained from borders of lesions suspicious of leishmaniasis in 298 patients, stained with Giemsa and cultured in NNN and RPMI 1640 media supplemented with 10% fetal colf serum. The promastigotes were then purified, counted and used as antigen in ELISA test. Results: Mass production of promastigotes was successful in 120 cases. L.major was identified as the causative agent of the disease in 100 cases and L.tropica in 8 patients. In 12 cases the results were inconclusive as the antigens reacted with either 2 or 3 specific monoclonal antibodies. Conclusion: L.major is the most common cause of leishmaniasis in Isfahan.