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Two predominant forms of cutaneous leishmaniosis are anthroponotic CL (ACL) and zoonotic CL (ZCL) caused by Leishmania (L.) tropica and L. major in Iran and many countries, respectively. Since differential gene expression play an important role in outcome of the infection, we compared relative gene expression value of pyruvate kinase (PyrK) and tryparedoxin peroxidase (TryP) in metacyclic forms of Iranian isolates of L. major and L. tropica. Clinical isolates of CL patients were sampled in endemic foci of Iran and identified by PCR-RFLP. Then, we employed real-time PCR to evaluation of the expression level of PyrK and Tryp genes in L. major and L. tropica. By this comparison, up-regulation of PyrK and Tryp genes was observed in metacyclic stage. Moreover, the average mRNA expression of PyrK and Tryp genes in L. major was 1.69 and 3.72 folds of its expression in L. tropica isolates. The results of this study could open the new window for further investigations of the correspondence between parasite gene expression level and disease pathology. Species-specific parasite factors contributing to virulence and pathogenicity in the host may be mostly due to the some of the differential regulation of conserved genes between species.