A total of 156 isolates of Leishmania from patients with cutaneous leishmaniasis and one isolate from gerbil were characterized using standard monoclonal antibodies (mAb) in ELISA and IFA test systems. The geographic distribution of the isolates was 30 isolates in Shiraz, 28 in Kerman and 98 in Tehran. A total of 63, 72, and three Leishmania promastigote isolates preferentially reacted with anti- Leishmania tropica mAb (A11), anti- Leishmania major mAb (T1) and anti- Leishmania infantum mAb (D2), respectively. Of the three isolates which reacted with anti- L. infantum mAb, two were dermotropic strains of L. infantum and one was the organism isolated from gerbil. Mixed reactions with the monoclonal antibodies were also observed as six isolates reacted with both anti- L. tropica and anti- L. major mAbs, five reacted with anti- L. tropica and anti- L. infantum mAbs and two with anti- L. major and anti- L. infantum mAbs. The monoclonal antibodies used did not react with five of the isolates. The present investigation has identified the organisms from the known and newly emerged foci of cutaneous leishmaniasis in Iran.