Phylogenetic analysis of the 16S ribosomal gene initial region is used to identify Leptospira isolates at the species level from clinical samples. Unfortunately, this method cannot differentiate between some intermediates and saprophytic species.We used comparative genomic analysis between 35 Leptospira species to find new molecular targets for Leptospira species identification.We proposed the use of the rpoC gene, encoding the DNA-directed RNA polymerase β-subunit, for identifying 35 Leptospira species.The rpoC gene can be a molecular target to identify the main species of the Leptospira genus directly from clinical samples.