Serological identification of Leptospira ssp isolates is difficult to achieve. Thus, molecular testing may be of great interest thanks to its high discrimination power, reproducibility and easy interpretation.To implement and validate conventional and multiplex PCR methods (using primers directed against lipl32 and secY/flaB genes, respectively). To assess the capacity of PCR methods to identify pathogenic and saprophytic species of Leptospira ssp.22 international reference strains and 12 colombian isolates were used. DNA was extracted with a commercial kit (Wizard). Specificity and sensitivity of both PCR methods were evaluated.The maximum dilution of DNA samples allowing the detection of Leptospira ssp was determined to be 1:10000 for the PCR lipL32 and 1:100/1:1000 for the multiplex PCR secY/flaB. Both PCR didn't detect DNA from microorganisms unrelated to Leptospira ssp. The lipL32 PCR specifically amplified a 423 bp fragment from all pathogenic Leptospira reference strains, while the secY/flaB PCR amplified both 285 bp (secY) and 793 bp (flaB) fragments from 18 reference strains. The lipL32 PCR detected 7/12 colombian isolates, while secY/flaB PCR detected both secY and flaB genes from 6/12 isolates.Best results were obtained with the lipL32 PCR, which displayed a better sensitivity and a better capacity to detect different strains than the multiplex PCR. The secY primers showed a poor specificity to pathogenic species and a poor sensitivity. Thus, lipL32 primers show high potential for molecular diagnosis of Leptospira spp in clinical and environmental samples.