Leptospirosis is a bacterial zoonotic disease caused by spirochetes of the genus Leptospira. The direct method of diagnosis of Leptospirosis has been so far by culture isolation but isolation rate of the microorganism from clinical specimens is low. The additional conventional method is the detection of antibodies which is done by ELISA, that fails to identify the infecting serovar. MAT is used as the gold standard, but it can only confirm the disease at a later acute phase because anti-Leptospira antibodies usually become measurable only 5 to 7 days after onset of illness. Thus, serology does not contribute to early diagnosis of Leptospirosis. To overcome these limitations, we developed PCR assay targeting ccmF gene of Leptospires using in-house designed RGf and RGr primers, with a product size of 910bp. The protocols were standardized. PCR could detect the target bacterial gene without any ambiguity. This simple, cost-effective and rapid method can be applied to detect causative Leptospira interrogans serovars from the isolates.__ampersandsignnbsp;