Rapid diagnosis of leptospirosis, through culture and/or serology, can be difficult without proper expertise and is often delayed because of the length of time required to obtain results. In this study, we developed a real-time polymerase chain reaction (PCR) assay using a TaqMan probe targeting lipL32, which is present only in pathogenic Leptospira spp. Using Leptospira interrogans serovar Icterohaemorrhagiae DNA, the lower limit of detection was found to be 20 genomic equivalents/reaction with a 95% cutoff value. The assay detected pathogenic Leptospira strains, but not intermediately pathogenic or nonpathogenic strains. When testing the assay on spiked clinical specimens, whole blood and plasma were better specimens for detecting the same initial number of leptospires compared with serum from clotted and centrifuged blood. Leptospira spiked at the same concentration was better detected in centrifuged urine. This real-time PCR assay with high specificity and sensitivity may prove to be a rapid method for diagnosing acute leptospirosis.