Introduction: Lishmania is a protozoan parasite that cause visceral and cutaneous disease in man. This parasite shows two forms in its life cycle, amastigote in reticulo-endothelial cell of mammalian host and, promastigote in culture media and sand fly vector. Like other kinetoplastide, leishmania has an extra chromosomal DNA with unique topological and characteristic that called kDNA. The organism is responsible of different clinical manifestation in man, therefore, identification of different species and strains was always demanded. Identification and characterization of leishmania depend on geographical disteribution, clinical manifestation, monoclonal antibody, serological methods, isoenzyme pattern and others. In last decade molecular methods was developed for these aims. DNA is stable through its life cycle and kDNA organized in two different molecules, minicircles has 0.5-2.9 kb with heterogeneity in sequences and variable in number, and maxicircles with 20-50 kb size that codes for enzymes and cofactors of mitochonderial genes. Materials and Methods: KDNA minicircle of leishmania major was extracted and DNA library was constructed by BamHI enzyme in pBluescript plasmid. After transformation, recombinant plasmid was screened by a-complementation test on X-gal and IPTG - treated agar plates. Results: Two clone of leishmania major kDNA in BamHI site of pBluescript plasmid were identifed. Ribiperobe syntesized by run off synthesis method pBluescript plasmid. To define probe specificity. They were examined with inserted DNA and total kDNA through dot blot and southern blot hybrydization by IRI-m2 RNA probe. IRI-m2 was hybridized with L. major with high specificity and lesser specifity with L. infantum. Conclusion: This sequence is suitable for PCR primer design and use for differentional of L. major and L. donovani by L. tropica.