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Introduction: Visceral leishmaniasis (VL) or Kala-azar is an important parasitic diseases caused by protozoan parasites of the genus Leishmania including L. donovani, L. infantum. Epidemiological evidences show that VL is frequently common in some areas of Jahrom district in Fars Province. This study aimed to characterize the causative agent of VL in this region. Materials and Methods: Bone marrow sample was obtained from 16 VL patients from Pediatric hospital in Jahrom town. DNA was extracted from the microscopic slides and checked by specific PCR to find out the species of the parasite. To do it, a segment of minicirclekinetoplast DNA was amplified, using LINR4 and LIN17 primers. Products of PCR were evaluated by electrophoresis, using 1.5% agarose. Results: Anti-leishmania antibody was detected in all patients by IFAT. Examination of bone marrow smears demonstrated numerous amastigotes of Leishmania (Leishman bodies) in the samples. All the samples produced a 720 bp band in PCR assay. The isolates were compared with reference strains and revealed that all the isolates are L. infantum. Conclusion: Our findings showed that species characterization with one-step PCR by LIN17 and LINR4 is simple and fast it was also confirmed that the causative agent of VL in Jahrom is L. infantum.