Leptospirosis, a globally prevalent zoonosis caused by pathogenic and intermediate Leptospira species, poses significant
threats to public health and livestock industries. Despite its substantial impact, knowledge gaps persist regarding the prevalence
and genetic diversity of Leptospira strains in many regions, including South America. This study aimed to characterize
a diverse collection of Leptospira strains isolated from various sources in Colombia to enhance our understanding of
the genetic diversity within this genus. Using a tiered approach combining conventional and genomic methods, we genotyped
55 isolates from various sources using 16S rRNA and rpoB gene sequencing, DNA ribotyping, and Multiple-Locus
Variable-Number Tandem Repeat Analysis (MLVA). Most isolates were classified into phylogenetic groups containing
pathogenic and intermediate strains of L. interrogans and L. wolffii, respectively, which was corroborated by ribotyping
and MLVA. Whole-genome sequencing of selected strains revealed distinct genomic characteristics compared to related
strains. Pan-genome analysis identified strain-specific genes, primarily hypothetical, while virulence factor analysis distinguished
species-specific patterns. Furthermore, CRISPR-Cas system analysis uncovered genetic variations among the
isolates. This study provides a framework for understanding Leptospira genetic diversity in Colombia and its potential
implications on human and animal health. Our findings highlight the need for improved diagnostic methods and surveillance
strategies that encompass both pathogenic and intermediate Leptospira species, which could significantly impact
public health policies and veterinary practices in the region.