Diagnosis of leptospirosis is currently based on serological tests detecting antibodies against the spirochete. The standard method is the microscopic agglutination test (MAT), which is serovar-specific, requires a period of antibody development, and increases the risk of exposure to viable organisms. Therefore, the present study aimed to develop a rapid, sensitive, specific, and safe method based on the PCR technique to detect pathogenic Leptospira in urine samples. Nested PCR using two sets of primers, external and internal primers, were shown to specifically amplify 16S rRNA target of patho-genic Leptospira. No amplification was observed when DNA from non-pathogenic Leptospira and other non-Leptospira bacteria were used as DNA templates. The method was able to detect as few as 10 leptospires in urine. Therefore, nested PCR approach may be a useful tool for prompt and definitive diagnosis of leptospirosis.