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<p><strong>Background</strong>: Leishmaniasis is a major medical health problem and distributes in nearly half of 31 provinces of Iran. We aimed to identify cutaneous and visceral <em>Leishmania</em> spp. isolated from infected humans and domestic dogs in various regions of Iran, 2010‒2013.<strong><br />Methods: </strong>DNA was extracted from 108 lesion exudate samples of suspected patients to cutaneous leishmaniasis and nine liver and spleen aspirates of infected dogs cultured in RPMI-1640 and amplified using partial sequence of ITS1 gene. The PCR amplicons were digested using <em>Hae</em>III endonuclease enzyme and used in restriction fragment length polymorphism (RFLP) assay. Then, 48 amplicons representing various hosts were sequenced and compared to se­quences from GenBank databases using BLAST.<strong><br />Results:</strong> PCR-RFLP analysis showed that 60 and 48 CL patients were infected by <em>Leishmania tropica</em> and <em>L. major</em>, respectively. From nine canine visceral leishmaniasis (CVL) isolates, eight isolates were identified as <em>L. infantum</em> and one as <em>L. tropica</em>. The greatest similarity of 95.7% in ITS1 region was seen between <em>L. infantum</em> and <em>L. major</em>. Furthermore, the lowest similarity with 65.7% was seen between <em>L. tropica</em> and <em>L. major</em>. Intra-species comparison of ITS1 region in <em>L. infantum, L. major </em>and<em> L. tropica</em> isolates were showed 100%, 98.2% and 72.4 % similarities, respectively.<strong><br />Conclusion:</strong> PCR-RFLP based on ITS1 region is an appropriate method to distinguish three <em>Leishmania</em> spp. of <em>L. major</em>, <em>L. tropica,</em> and <em>L. infantum</em>. In intra-species comparison of ITS1 region, genotypic variations showed that <em>L. tropica</em> isolates were more heterogeneous than <em>L. major</em> and <em>L. infantum</em> isolates.</p>