Background and objective : Cutaneous leishmaniasis is an endemic diseases with important public health in Iran. The cinical signs of disease were seen as dry and wet forms. It is essential to distinguish leishmania species in every area for designing control of diseases. The morphology of leishman bodies in each speiecis are very similar and need to sensitive diagnostic method such as PCR to differntiate of them. The present study,a molecular study has been done to identify of leishmania species in Mashhad area from autumn in 2011 to summer in 2013. Methods: Firstly, tissue smears were collected from the lesion of one hundred patients and from the ulcer of 25 paitents were cultured in NNN nutrified with RPMI . The positive samples (tissue smears and culture) were used for DNA extraction and PCR. PCR methods were used in two steps. Firstly a sensitive PCR was used to detect the leishmania genus and secondly , the positive samples were examined with species specific semi-nested - PCR Results: In first step of PCR, all of sampled were positive for Leishmania spp and in second step Leishmania tropica and L.major were detected in 94% and 6% in positive –PCR amplicon , respectively. Conclusion: Based on the results, Leishmania tropica is more prevalent than L.major in Mashhad area