Leptospirosis is an acute infectious, systemic and septisemic disease which had recent outbreaks in some parts of Iran especially in north provinces. Rapid detection is a critical step for treatment and control of this disease. In this research a PCR based method was evaluated for detection of Iranian local endemic serovars. All reported endemic serovars of Leptospira including Leptospira grippotyphosa, Leptospira canicola, Leptospira sejreo hardjo, Leptospira pomona and Leptospira icterohaemorrhagiae which at present are used for Leptospira micro agglutination test (LMAT) in Iran, were subjected to this PCR. Standard representative serovars from ATCC studied in parallel to local serovars. DNA extracted by phenol-chloroform-isoamyl alcohol precipitation. After optimization, the sensitivity of PCR was about 1 fg equal to DNA of 1 Leptospira. All studied serovars including non pathogenic L. biflexa produced the reported 285 bp fragment. Restriction analysis with MboI confirmed the PCR product accuracy. In case of L. biflexa the pattern was completely different from the pathogenic ones. None of near matching bacteria had product in this method. This system was able to detect the existence of Leptospira DNA in all of studied LMAT positive serums.