BACKGROUND AND OBJECTIVES: Leptospirosis is a zoonotic disease caused by pathogenic serovars. The genus cannot differentiated by conventional techniques. However, identity determination of pathogenic serovar is precious of public health problems and epidemiological studies. Pulsed-field gel electrophoresis facilitates rapid identification of to the serovar levels. MATERIALS AND METHODS: In this study, we employed PFGE to evaluate 28 isolates, with animal, human and environmental origin, obtained from Razi Vaccine and Serum Research Institute of Karaj, Iran. PFGE patterns of 28 serovars were generated using the I restriction enzyme in comparison with the lambda ladder. RESULTS: Out of 28 serovars evaluated, we identified 22 different pulsed types, designated P1-P22. Out of 22 pulse groups, 3 were found to be a common type, but others were a single Type. Groups consisting of the common type were P3, P9, P14, and P16. The results showed that the discriminatory index of PFGE by I enzyme was 0.99, demonstrating heterogeneous differentiation among serovar members. CONCLUSION: The PFGE methodology used in this study showed excellent interlaboratory report usability, rapid, reliable, enabling standardization and data sharing between laboratories.