To examine the genetic diversity ofLeishmania major, 100 Giemsa-stained positive slides were collected from endemic foci of Iran (Northeast, Central, and Southwest provinces) over two consecutive years during 2019–2021. TheLeishmaniaITS-rDNA gene was amplified andLeishmaniasp. was recognized by PCR-RFLP and sequencing. In addition, 178 registered ITS-rDNA sequences from other geographical regions of Iran were retrieved from GenBank, including different host species (human, sandfly and rodent). A total of 40 new haplotypes were discovered using the ITS-rDNA sequence analysis. IR29 (20.6%) and IR34 (61%) were the two most common haplotypes, represented by a star-like feature in the overall population. Analysis of the molecular variance test revealed low genetic diversity ofL.majorin human cases (Haplotype diversity; 0.341), rodent (Hd; 0.387) and sandfly (Hd; 0.390) sequences. The lowest genetic diversity ofL.majorwas observed in Southwest/Southeast Iran (Hd: 0.104–0.286). The statisticallyFst value indicated thatL.majoris not genetically differentiated between geographic regions of Iran, except for the Northeast-Southwest (Fst: 0.29055) and Central-Southwest (Fst: 0.30294) population pairs. The current study as the first investigation discloses new perspectives for further evaluation in the identification local transmission paradigms and initiating effective prevention strategies.