Leptospirosis is an emerging zoonotic disease. The differential diagnosis of this disease is difficult due to the varied symptoms which may result in a missed or delayed diagnosis. In order to overcome these limitations, a real-time PCR assay was optimized using a TaqMan probe and primer target on 16s RNA of Leptospira. 15 pathogenic and 1 non-pathogenic Leptospiral serovars and some other bacteria strains were tested. The assay was specific for Leptospira and may discriminate pathogenic and nonpathogenic species. Applied this assay to detect Leptospiral DNA from pig kidney specimens shown that there were 16.1 percent of specimens collected in slaughter house in Khanh Hoa and Kon Tum provinces to be positive.