Purpose: This study aimed to identify the causative leishmania species of cutaneous leishmaniasis (CL) in Al-Diwaniyah Governorate, a central regin in Iraq using molecular technique. Methods: Twenty clinically suspected cases were collected from patients who visited Al-Diwaniyah Hospital during the period between January and April 2024. Initial diagnosis was performed using Giemsa stained microscopy and parasite culture on RPMI-1640 medium. Molecular characterization was performed using polymerase chain reaction (PCR) amplification targeting the mitochondrial cytochrome b (Cyt b) gene, followed by DNA sequencing and BLAST analysis to determine species identity and genetic relationships. Results: All samples were confirmed to be infected with cutaneous leishmaniasis. Molecular analysis of the cytochrome b gene revealed that the isolates were Leishmania tropica. With limited nucleotide variation, it did reveal minor amino acid differences, leading to the classification of the isolates into three groups. Conclusion: The results confirm that Leishmania tropica is the predominant causative agent of cutaneous leishmaniasis in the study area. The study highlights the importance of molecular diagnostics for accurate identification of the parasite.