We describe a case of visceral leishmaniasis (VL) due to Leishmania tropica in a 50-year-old Iranian man lived in a VL-endemic area in southwest of Iran. The patient presented with a 3-month history of fever and splenomegaly. Clinical signs and serological findings were suggestive of VL. Spleen biopsy was taken from the patient and intracellular forms of Leishmania amastigotes was seen in Giemsa stained smears. The patient was treated with pentavalent antimonial compound with complete resolution of his systemic signs and symptoms. DNA was extracted from the microscopic slides of the spleen biopsy and the nagt (N-Acetylglucosamine-1-Phosphate Transferase) gene of Leishmania was PCR-amplified. Sequence analysis of the PCR product demonstrated that the case has 99% identity with those of available sequences of L. tropica. Intra-species variation within isolate was 0-0.1%; whereas, inter-species differences of the isolate with those of L. major and L. infantum was significantly higher.