Objective: To determine an algorithm for molecular diagnosis of visceral leishmaniasis (VL) by kinetoplast DNA (kDNA) (RV1/ RV2) and intal transcriber spacer (ITS1) (LITSR/L5.8S) polymerase chain reaction (PCR),complemented by ITS1 PCR restriction fragment length polymorphism (RFLP),using peripheral blood or bone marrow aspirate from patients with suspected VL.Methods: Biological samples were submitted to the gold standard for the diagnosis of VL and molecular diagnosis represented by ITS 1 PCR,kDNA PCR,and ITS 1 PCR RFLP.The samples were obtained from seven groups: group Ⅰ,82 samples from patients with confirmed VL;group Ⅱ,16 samples from patients under treatment for VL;grouplⅢ,14 samples from dogs with canine visceral leishmaniasis (CVL);groupⅣ,a pool of six experimentally infected sandflies (Lutzomya longipalpis);group V,18 samples from patients with confirmed tegumentary leishmaniasis (TL) and groups Ⅵ and Ⅶ were from control groups without VL.Results: The following gold standard and molecular examination results were obtained for each of the seven groups: group Ⅰ : parasitologic and immunochromatographic tests showed a sensitivity of 76.3％ (61 of 80) and 68.8％ (55 of 80),respectively,and a sensitivity of 97.6％ (80 of 82) and 92.7％ (76 of 82) by ITS1 and kDNA PCR,respectively.After ITS 1 PCR RFLP (Hae Ⅲ) analysis of the 80 positive samples,52.5％ (42 of 80) generated three fragments of 180,70,and 50 bp,corresponding to the patt of Leishmania infantum infantum;group Ⅱ : negative for the parasitologic methods and positive for IrK39 (100％,16 of 16),presented 12.5％ (2 of 16) of positivity by ITS1 PCR and 25.0％ (4 of 16) by kDNA PCR;groupⅢ: positive in the parasitologic and serologic tests (100％,14 of 14),presented 85.7％(12 of 14) of positivity by ITS1 PCR and kDNA PCR.ITS 1 PCR RFLP showed that 83.3％ (10 of 12) of the canine samples contained parasites with profiles similar to L.infantum;groupⅣpresented amplifications by ITS 1 PCR and kDNA PCR.ITS 1 PCR products were analyzed by RFLP,generating a profile similar to that of L.infantum;group V: positive in the parasitologic examination (100％,18 of 18),presented 72.2％ (13 of 18) of the samples by ITS1 PCR positive.A total of 69.2％ (9 of 13) showed profiles corresponding to a Viannia complex by ITS1 PCR RFLP;and groupⅥ and group Ⅶ were negative by ITS 1 and kDNA molecular tests.Comparing the molecular results with the parasitologic and serologic diagnosis from group Ⅰ,almost perfect agreement was found (κ both＞0.80,P＜0.001).ITS1 and RV1/RV2 PCR detected 90.2％ (74 of 82) of the samples.Two samples positive by RV1/RV2 were negative by LITSR/L5.8S,and six samples positive by LITSR/L5.8S were negative by RV1/RV2.Therefore,these two systems complemented each other;they diagnosed 100％ of the samples as belonging to the Leishmania genus.Conclusions: We suggest an algorithm for the molecular diagnosis of VL,which must consider previous parasitologic and serologic (immunochromatographic) diagnoses,and should combine kDNA and ITS1 to determine the Leishmania subgenus using RFLP as a complement method to define the L.infantum species.