Background Leptospirosis is a worldwide zoonosis that is endemic in tropical areas. The species Leptospira interrogans is the primary agent in human infections, but other pathogenic species, such as L. kirschner and L. borgpetersenii, are also associated with human leptospirosis.

Methods and Findings In this study, an unsupervised high resolution melting (HRM) analysis of the products that were amplified with five pairs of primers lfb1 F/R, G1/G2, VNTR-4Bis, VNTR-Lb4 and VNTR-Lb5 facilitated an accurate species classfication of Leptospira reference strains from New Caledonia Institute Pastéur. Next, the genotypes at the subspecies level was identificated by using method with LightCycler®480 instrument and the High Resolution Melting Master kit (04909631001). LightCycler®480 Gene Scanning Software was used to perform a futher analysis results.

Conclusions This new HRM method enabled the identification of Leptospira strains at the species and subspecies levels and support the direct genotyping of Leptospira in biological samples without requiring cultures.