The performance of PCR to detect Leishmania kDNA in serum for the diagnosis of visceral leishmaniasis (VL) was assessed in serum samples from 65 patients with VL, 17 non-infected individuals and 17 patients with other febrile hepatosplenic diseases. Serum PCR showed a sensitivity of 85%, specificity of 100% and efficiency of 90%. The sensitivity values obtained for blood PCR (97%) and rK39 ELISA (95%) were significantly higher (P=0.01) than the values observed for L. chagasi ELISA (88%) and serum PCR (85%), whilst no difference was observed among the specificity rates obtained with rK39 ELISA (94%; P=0.47) and L. chagasi ELISA (85%; P=0.06). This work suggests that the use of serum samples may be an alternative for the diagnosis of VL when peripheral blood samples are not available or require significant operational efforts.