Leptospira is a pathogenic bacteria, which causes Leptospirosis in humans and animals. The genome sequence of Leptospira interrogans Lai (Pathogenic) and Leptospira biflexa Patoc (Non-Pathogenic) were retrieved and examined for the presence of Short Sequence Repeats (SSR) (n=1,2,3)in Chromosome I and Chromosome II. SSRs or microsatellites extensively exist in genomes of prokaryotes and eukaryotes. Simple sequence repeats are the genetic loci where the bases are tandemly repeated for varying number of times. Comparative genome analysis will provide a better understanding in the identification of the evolutionary relationship between pathogenic and non-pathogenic species. SSRs in genome sequences (pathogenic and non- pathogenic) were found using ‘R’ script, which was implemented in R packages. It was observed that the pathogenic sequence contains a number of tandem repeats in both the chromosomes. Meanwhile, the occurrence of C/G or G/C has more difference in their frequencies between pathogenic and non-pathogenic sequences. In both the chromosomes, dinucleotide repeats are frequent, but in the case of TC in both the chromosomes and GA in chromosome II are found to be less significant. Additionally, trinucleotide repeats are longer in pathogenic, whereas in non-pathogenic they are shorter. The statistical analysis of the microsatellites in both the sequences indicates the highly significant pattern of nucleotide repeats. The more number of genes in the pathogenic species may be acquired to the virulence in their course of evolution. This work partially suggests that SSRs plays a major role in genetic diversity, gene evolution and also in understanding the genomic instability.