Background: As a neglected disease, cutaneous leishmaniasis renders considerable incidence rates. Eastern Mediterranean countries such as Iran are endemic for this infection. Objectives: Herein, we determined the species/strains of the causative agents of cutaneous leishmaniasis in Dasht-e-Azadegan, Khuzestan, Iran, using PCR-RFLP in 2016. Methods: We microscopically examined 80 smear slides from suspected patients referring to health centers of the city. After DNA extraction from slide materials, the amplification of the ITS1 fragment was done using the LITSR and L5. 8S primer pair for Leishmania molecular detection. Subsequently, haeiii (species level), TaqI (strain level), DpnI, andHpaII (mutation analysis) digestionwasexerted based on the RFLP method. Results: Microscopic examination revealed the amastigote forms of Leishmania in all 80 samples. A 350 bp band was amplified by ITS1-PCR, which confirmed the infection at the molecular level. Following haeiii digestion, 150 and 200 bp fragments were produced indicating Leishmania major. Also, TaqI digestion rendered 130 and 200 bp bands suggesting the A1 strain. Moreover, no mutations were detected in the genome of the identified L. major A1 strains by DpnI (140 and 200 bp bands) and HpaII (no digestion) digestion. Conclusions: The genotypic heterogeneity of Leishmania species is of utmost importance for better treatment choices, appropriate diagnosis, and preventive measures. Future work in the area should address the parasite strains in alternative vector/reservoir hosts.