Aims: Leptospirosis is an infectious disease that is endemic to many tropical regions. Large epidemics usually happen after heavy rainfall and flooding. This potentially fatal zoonosis is caused by pathogenic bacteria belonging to the genus Leptospira. Leptospirosis can be diagnosed using specific biomarkers such as target genes and virulence indicators that are well preserved across various Leptospira spp., including those that are prevalent in clinical samples and in the environment. To date, several pathogenicity-determinant genes, including lipL32 and lipL41, have been described and used for diagnosing leptospirosis. However, prevalence of these genes in leptospiral strains is unclear. Methodology and results: In the present study, we assessed the distribution of eight pathogenicity-determinant genes in reference Leptospira strains and environmental isolates in Malaysia, by polymerase chain reaction (PCR). We found that only lipL32 and ligB were consistently expressed in all pathogenic Leptospira strains compared with the other tested genes. Moreover, our results suggested that the use of lipL41, lipL21, ompL1, lfb1, ligA, and ligC as biomarkers could incorrectly misdetect pathogenic Leptospira strains present in the environment. Conclusion: Thus, our results suggest that the pathogenicity-determinant genes lipL32 and ligB can be used as biomarkers for detection pathogenic Leptospira