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Leptospirosis is a common zoonotic disease in tropical and subtropical countries. It is considered an emerging disease in Malaysia and is a notifiable disease. This study was conducted to characterize Malaysian isolates from human, animal and environmental samples via MLST and <i>rrs2</i> sequencing in an attempt to develop a Malaysian genotypic database. An existing polymerase chain reaction (PCR)-based MLST scheme was performed to facilitate subsequent sequencing. Out of 46 extracted DNA, 36 had complete MLST profiles whereby all six genes were amplified and sequenced. Most of the pathogenic <i>Leptospira</i> genotypes with full MLST profiles were <i>L. interrogans</i> serogroup Bataviae (<i>n</i> = 17), followed by <i>L. borgpetersenii</i> serogroup Javanica (<i>n</i> = 9), <i>L. interrogans</i> serogroup Sejroe (<i>n</i> = 2), <i>L. interrogans</i> serogroup Australis (<i>n</i> = 2), <i>L. kirschneri</i> (<i>n</i> = 2), <i>L. interrogans</i> serogroup Grippotyphosa (<i>n</i> = 1) and <i>L. interrogans</i> serogroup Pyrogenes (<i>n</i> = 3). Two samples (R3_SER/17 and R4_SER/17) were not closely related with any of the reference strains. For the samples with incomplete MLST profiles, leptospiral speciation was conducted through <i>rrs2</i> analysis, in which four samples were identified as <i>L. borgpetersenii</i>, five samples were closely related to <i>L. kmetyi</i> and one sample was known as <i>L. yasudae</i>. This study shows that molecular approaches that combine both MLST and <i>rrs2</i> sequencing have great potential in the comprehensive characterization of pathogenic <i>Leptospira</i> because they can be performed directly from cultured and clinical samples.