A urine-polymerase chain reaction (PCR) assay was validated for diagnosis of human visceral leishmaniasis (VL), taking advantage of the accessibility of urine samples. Leishmania infantum DNA presence was examined in 17 urine samples from 17 patients with VL during a clinical episode and in 55 urine samples from 17 patients with VL monitored after treatment at different intervals. Fifty-nine urine samples from 59 controls with no history of VL were also studied. The urine-PCR test was positive in 15/17 samples obtained during the episode (sensitivity, 88%). None of the controls tested were urine-PCR positive (specificity, 100%). During the monitoring period, 25% of the samples gave a positive urine-PCR. Results were compared with other diagnostic methods, such as urine antigen detection and peripheral blood-PCR and culture, with good concordance during the clinical episode and differences in the follow-up period. This study suggests that urine-PCR is sensitive for diagnosis and may be useful to monitor treatment efficacy.