Iran is one of the six countries with the most cutaneous leishmaniasis (CL) patients. Understanding better the genotypes of the parasite population in relation to geography and climate is critical to achieving better CL control. We aimed to characterise the population structure of , the cause of anthroponotic cutaneous leishmaniasis (ACL), from important foci in southeast (Bam and Kerman) and southwest (Shiraz) Iran. A total of 39 isolates from ACL patients from southeast (Bam 14, Kerman 12) and southwest (Shiraz 13) Iran were analysed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of the kinetoplast DNA (kDNA) using restriction enzymes I () and I. 37 genotypes were identified among south Iran isolates. The unweighted pair group method with arithmetic mean (UPGMA) tree obtained from the banding patterns of I digested kDNA RFLP distinguished southeast from and southwest isolates with some subclustering but the I derived tree showed greater discrimination with greater subclustering and divergence of the two foci of southeast region but with some overlapping. Although a monophyletic structure has been defined for southeast , isolates from two foci of southeast Iran were partly discriminated in the current study.