Zoonotic cutaneous leishmaniasis (ZCL) is an expanding disease and a public health issue in Iran. In the present study, rate of natural infection of rodent populations with Leishmania was investigated in six endemic foci including 28 villages in Golestan, Esfahan, Yazd, Fars, Khuzestan and Ilam provinces. A total of 593 rodents were captured and identified as Rhombomys opimus (n = 325), Meriones libycus (n = 171), Meriones persicus (n = 27), Tatera indica (n = 37), Nesokia indica (n = 12), Rattus rattus (n = 13) and Mus musculus (n = 8). Microscopic examinations of Giemsa-stained smears showed that 108 out of 593 (18.2%) rodents were infected with Leishmania spp., whereas infection of 186 out of 593 (31.4%) rodents with Leishmania was then confirmed by ITS1-PCR. The highest rate of infection was found in R. opimus (prevalence of 35%) and M. libycus (31%). Based on Restriction Fragment Length Polymorphism (RFLP), 145 (78%) of 186 samples detected as Leishmania DNA were identified as L. major, 8 (4%) samples as L. turanica and 33 (18%) as mixed infection (L. major and L. turanica). Samples from infected rodents were inoculated subcutaneously at tail base of BALB/c mice. In 35 of them, nodules and ulcers containing amastigotes appeared at the inoculation site. The samples prepared from infected rodents were cultured in NNN medium and only two samples werepositive. Rhombomys opimus, M. libycus, M. persicus, T. indica and N. indica were confirmed as reservoir hosts of ZCL in the studied regions. Leishmania major infection was usually accompanied L. turanica in naturally infected gerbils (R. opimus and M. libycus) in Golestan, Esfahan and Fars provinces.