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• Among the 181 gerbils captured, 48.6% were Leishmania spp. positive upon microscopic examination. • Leishmania infection was observed in 89.5% of the gerbils using nested-PCR. • Single or mixed infections of L. major with L. gerbilli or L. turanica were detected in 28.4% gerbils. • Homology investigation using NCBI blast against Genbank database sequences revealed 92.45–100% homology between samples and L. major in the Genbank sequences using ITS2-rDNA. Rodents from the subfamily Gerbillinae are the principal reservoir of cutaneous leishmaniasis (CL) caused by Leishmania major in the center and northeast of Iran. This study was conducted to determine both naturally occurring Leishmania infection rates and the distribution of Leishmania species in the central parts of Iran during 2019–2020. In this regard, presence of Leishmania parasites were confirmed by microscopic examination and the species were identified by nested-PCR using the Internal Transcribed Spacer2- Ribosomal DNA (ITS2-rDNA). Finally, some samples were sequenced and, the blast analysis of L. major samples, showed a 92.45–100% homology to the L. major sequence. Of the 181 wild gerbils collected (Rhombomys opimus =157 and Meriones lybicus =24), 88 (48.6%) tested positive for Leishmania sp. by microscopic examination whereas 162 (89.5%) were positive by nested-PCR. Of the 162 infected gerbils, 103 showed single strain infections (30 L. major, 28 L. gerbilli and 45 L. turanica) , 43 showed dual infections with only the non-human species (L. gerbilli and L. turanica) , and 16 were mixed infections of L. major and L. turanica (n = 14) or L. gerbilli (n = 2). All single or mixed L. major infections were detected in gerbils from areas with reports of human CL during the last decade. These findings suggest that Rhombomys opimus and Meriones libycus have a potential role in the maintenance of human and non-human transmission of Leishmania species in the CL foci. [ABSTRACT FROM AUTHOR]