Objective: To detect genetic variations among pathogenic Leptospira isolated from rats using 16S rRNA gen as chronometer. Methods: This is an observational study with cross sectional design. Rats samples were taken in Yogyakarta Special Region of Indonesia. Leptospira in the rats was detected by two methods i.e. real time PCR (qPCR) by using primers correspond to16S rRNA gene of Leptospira, and standard PCR by using different set of primer correspond to the 16S rRNA gene of Leptospira. The standard PCR amplicon then subjected for DNA sequencing. Analysis genetic variation was performed using MEGA 6.2. Software. Results: There were 99 DNA samples from rats included in this study. Detection of Leptospira by using qPCR revealed 25 samples positive for pathogenic Leptospira, while only 6 samples were able to be detected using standard PCR. The new primer set correspond to 16S rRNA gene was able to detect specifically pathogenic Leptospira in the rats. Sequencing analysis of 6 PCR amplicons showed that the Leptospira which infect the rats catched in Yogyakarta genetically close related with pathogenic Leptospira which were isolated from human, animal, rodents, and environment. Conclusions: It can be considered that rats are the most important vector and reservoir of Leptospira.