Abstract: Cutaneous leishmaniasis is a common skin disease caused by leishmania parasite. An accurate diagnosis of parasites species is possible using molecular techniques. This study was carried out to compare internal transcribed spacer (ITS1) and kinetoplast deoxyribonucleic acid (KDNA) genes for identifying Leishmania species by Polymerase Chain Reaction (PCR), furthermore, genetic diversity of isolates was studied. This research examined 130 patients who were suspected of cutaneous leishmaniasis and referred to Kashan's health centers from 2011-2014. After DNA extraction from serosity, PCR were performed using ITS1 and KDNA primers. Cutaneous Leishmaniasis was diagnosed by the observation of 320 bp band in the ITS1-PCR. The PCR products were digested with restriction enzyme HaeIII and then leishmania species were identified by patterns of enzymatic digestion. The diagnostic criteria of Cutaneous Leishmaniasis (CL) in KDNA-PCR were based on the observation of 760 and 650 bp for Leishmaniasis tropica and Leishmaniasis major, respectively. Twelve isolates of leishmania were sequenced and the phylogenetic tree was traced using the results of sequencing by Mega 4 software. Out of 130 suspected patients to CL, 70 (53.8%) and 98 (75.4%) isolates were positive by Restriction Fragment Length Polymorphism (RFLP) of ITS1 and KDNA, respectively. Using ITS1 PCR, 60 samples (85.7%) and 10 samples (14.3%) were identified as L. tropica and L. major, respectively, ITS1-PCR had 25.3% false negative, compare to microscopy. While, microscopy had false negative in 13 cases compare to KDNA-PCR. Due to the lower sensitivity of the PCR-RFLP of ITS1, KDNA-PCR is recommended for diagnosis of CL. The L. tropica and L. major are the causative agents of CL.