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Introduction: Leishmaniasis is one of the six important tropical diseases so that World Health Organization have recommended and supported to study its different aspect. Leishmaniasis is one of endemic parasitic disease in Iran and Isfahan province is one of the focus of Leishmaniasis in Iran. Material & Methods: Reservoir hosts of ZCL were captured by live trap. Rodents species were identified. Smear of each ear were prepared by scratching ear. Serous from rodent ears were isolated then, inoculated to NNN, injected to susceptible animal. Slides were prepared to find Leishmania using microscope. DNAs were extracted by ISH Horovize method and gene was amplified by Nested PCR and directly were sequenced. Finding: 50 rodents were trapped from 4 study regions. 40 Rhombomis opimus and 10 Meriones lybicus were traped. Leishmania infections were found(57%) in R. opimus and M. lybicus using conventional methods( direct smear, inoculation in Balb/C and in NNN medium). Detection of Leishmania major in those rodents was confirmed molecularly(%54). Sequencing results of samples confirmed two diferrent haplotypes of L. major in Isfahan rodents. Discussion & Conclusion: Based on finding, abundant of R. opimus and high Leishmania infection in this rodent it seem that R. opimus is the main reservoir of Leishmania major in Isfahan province followed by M. libycus as a second reservoir host of ZCL.