Leishmania major is the causative agent of zoonotic cutaneous leishmaniasis (ZCL) in which gerbils are the reservoir host. ZCL is of great public health importance in Iran. In the current investigation, nested polymerase chain reaction (PCR) protocols were used to amplify a region of the ribosomal RNA amplicon of Leishmania (ITS1-5.8S rRNA gene). The PCR assays detected L. major in three rodent species: Rhombomis opimus, Meriones lybicus and, for first time, Meriones persicus. L. major parasite was found in Natanz, Isfahan Province in the center of Iran in a focus of rural zoonotic cutaneous leishmaniasis. Four L. major infections were detected in R. opimus species, three in M. Lybicus, and two in M. persicus. All nine rodent infections of L. major were found to be the same haplotype based on the PCR detection and sequencing of parasite ITS-ribosomal DNA gene. In addition, also for the first time, the nested PCR assays detected Leishmania tropica only in one M. persicus. Allied to studies in country, the new findings mean that past conclusions about the reservoir of L. major in Iran must be treated with caution. Finding two Leishmania species in different rodent species as reservoir in Iran, therefore, careful molecular eco-epidemiological investigations will be an essential part of modeling the roles of different gerbil species in maintaining and spreading ZCL foci.