Genetic relationships among 25 isolates of Xanthomonas fragariae from diverse geographic regions were determined by three PCR methods that rely on different amplification priming strategies: random amplified polymorphic DNA (RAPD) PCR, repetitive extragenic palindromic (REP) PCR, and enterobacterial repetitive intergenic consensus (ERIC) PCR, The results of these assays are mutually consistent and indicate that pathogenic strains are very closely related to each other, RAPD, ERIC, and REP PCR assays identified nine, four, and two genotypes, respectively, within X, fragariae isolates, A single nonpathogenic isolate of X. fragariae was not distinguishable by these methods, The results of the PCR assays were also fully confirmed by physiological tests, There was no correlation between DNA amplification product patterns and geographic sites of isolation, suggesting that this bacterium has spread largely through exchange of infected plant germ plasm, Sequences identified through the RAPD assays were used to develop three primer pairs for standard PCR assays to identify X. fragariae. In addition, we developed a stringent multiplexed PCR assay to identify X. fragarine by simultaneously using the three independently derived sets of primers specific for pathogenic strains of the bacteria.