DNA polymorphism and variation in virulence of Xanthomonas axonopodis pv. manihotis (Xam), the causal agent of cassava bacterial blight, were studied within a pathogen population from Venezuela. Collections were made in several fields at different sites within an edaphoclimatic zone where cassava is a major crop. DNA polymorphism was assessed by RFLP analysis, using an Xam plasmidic DNA sequence (pthB) as a probe to determine the relatedness of 91 Venezuelan isolates. A high degree of polymorphism existed among the isolates, whether collected from the same or different fields. Based on a multiple correspondence analysis, the Xam population was distributed into eight clusters and no correlation was observed between genetic diversity and geographic origin. One set of haplotype strains representing the range of variability detected in Venezuela was further characterized by another RFLP analysis using two repetitive genomic probes (pBS6 and pBS8) to establish the usefulness of these probes and their complementarity with the pthB probe. Variation for virulence was observed in the Xam Venezuelan collection by inoculating a set of cassava cultivars with 28 isolates of the pathogen, each representing a haplotype. Understanding the genetic and pathogenic variation in the pathogen population is useful for designing cassava bacterial blight management strategies.