Leishmaniasis is one of the neglected tropical diseases, distributed across 89 countries in both the Old and New Worlds. Among the 54 identified species, 21 are known to be pathogenic to humans. Cutaneous leishmaniasis (CL) is primarily caused by and , while visceral leishmaniasis (VL) in Iran is caused by . Accurate detection and species identification of spp. are crucial for more effective treatment, epidemiology, and control strategies for the disease. Among the molecular targets recently used for detecting species, the heat-shock protein 70 () gene has proven to be highly suitable. This study aimed to establish and evaluate a SYBR Green real-time PCR targeting the gene to identify and differentiate three species: , , and in clinical specimens. A total of 219 microscopic smears, consisting of both positive and negative leishmaniasis cases diagnosed by microscopy, were subjected to DNA extraction and the real-time PCR assay designed in this study. Based on the analysis of the melting temperature (T) of the amplified target, 115 microscopy-positive smears were identified, comprising 70.4% , 23.5% , and 6.1% . All results were confirmed using a commercial diagnostic kit. Sanger sequencing of selected positive amplicons unequivocally confirmed the accuracy of this method in identifying and distinguishing the three species. The real-time PCR can be considered an effective method for detecting and identifying species from microscopic slides prepared from CL and VL cases in different regions of Iran.