| Summary: | Leptospirosis is an important worldwide zoonosis, and it has also been reported in Slovenia. The cultivation of Leptospira from human material is difficult. Despite that, we successfully isolated 12 human <i>Leptospira</i> strains isolated from patients between 2002 and 2020 and used various methods for the phenotypic and genotypic characterization of the strains, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) using our own MALDI-TOF data library, melting temperature analysis of the amplified <i>lfb1</i> gene, determination of <i>Leptospira</i> serogroups using rabbit immune sera, <i>NotI</i>-RFLP of the whole <i>Leptospira</i> genome, multilocus sequence typing (MLST) of seven housekeeping genes, and whole-genome sequencing (WGS)-based typing. We confirmed the presence of four pathogenic <i>Leptospira</i> species (<i>L. kirschneri</i>, <i>L. interrogans</i>, <i>L. borgpetersenii</i>, and <i>L. santarosai</i>) and three serogroups: Grippotyphosa, Icterohaemorrhagiae, and Sejroe. MALDI-TOF identified three of seven isolates at the species level and four isolates at the genus level. Serovars of 8 of the 10 strains were determined using <i>NotI</i>-RFLP. MLST showed that the clinical isolates belonged to sequence types ST17, ST110, and ST155. WGS confirmed the analysis of <i>Leptospira</i> strains using conventional methods. In addition, WGS provided better taxonomic resolution for isolate DDA 10944/10.
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