The usual target for sequence-based identification of Leptospira species is the 16S rRNA gene. However, because the 16S rRNA gene is not polymorphic enough, it is necessary to sequence a 1500 bp segment of this gene for accurate identification. Based on the alignment of previously determined rpoB of three Leptospira strains, we designed and tested a primer pair that enabled us to amplify and sequence a 600 bp segment of Leptospira rpoB. This segment was species-specific for the 16 species tested, but was unable to separate Leptospira interrogans serovars accurately. For the 11 L. interrogans serovars tested, only seven genotypes could be determined. We thus think that analysis of partial rpoB may be useful as an initial screening test for the identification of a new isolate of Leptospira and detection or identification of Leptospira in clinical or environmental samples, but not for serovar determination.