AIM: To develop a simple and accurate technique for the diagnosis of visceral leishmaniasis (VL) and identification of Leishmania pathogen. METHODS: Based on a set of oligonucleotide primers I and II designed by the authors with L. donovani specificity, a polymerase chain react ion (PCR ) was conducted to amplify a minicircle kDNA fragment (297 bp ) for identification and detection of L. donovani in the bone marrow , blood and serum samples from 22 confirmed VL patients and 4 bone marrow samples from 4 clinically diagnosed cases (59 samples collected from 26 cases). RESULTS: (1) The coincidence rate between PCR and bone marrow smear method was 96.2% (25/26) ; (2) The total positivity of PCR with the bone marrow , blood and serum was 95.4% (21/22) ; (3) The positive rate of PCR amplification with bone marrow , blood and serum specimens were 91.0% (20/22) , 68.8% (11/16)and 29.4% ( 5/17) , respectively. The controls included 15 bone marrow samples from 9 leukemia patients and 6 normal persons; 5 blood and 5 serum samples of normal persons. No amplification product was showed from all of the controls. CONCLUSION: The result shows that the diagnosis of visceral leishmaniasis based on detecting kDNA in blood by PCR amplification with primers I and II is promising.