ABSTRACT Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis, is a widespread disease that affects cassava (Manihot esculenta). We collected 238 X. axonopodis pv. manihotis strains by intensively sampling single fields in four edaphoclimatic zones (ECZs) in Colombia. DNA polymorphism of different X. axonopodis pv. manihotis populations was assessed by restriction fragment length polymorphism (RFLP) analyses, repetitive sequence-based polymerase chain reaction (rep-PCR), and amplified fragment length polymorphism (AFLP) assays. Genetic diversity, phenetic relationships among strains, and the coefficient of genetic differentiation were determined. All strains were tested for aggressiveness on the susceptible cassava cv. MCOL 1522. Strains were also tested for virulence on cassava differentials adapted to the strains' respective ECZs. Our study showed that the Colombian X. axonopodis pv. manihotis population has a high degree of genetic diversity. The hierarchical analysis of diversity showed genotypic differentiation at all levels, among ECZs, among fields within ECZs, and among strains within fields planted to several cassava genotypes. New RFLP haplotypes were detected, leading to the characterization of a new pathotype. Dendrograms from AFLP were more robust than those from RFLP data. A close association between the strains' geographical origin and DNA polymorphism was obtained using RFLP and AFLP data. We suggest that the host played a role in causing pathogen differentiation.