The delineation of bacterial species is presently done by direct DNA-DNArelatedness studies of whole genomes. It would be helpful to obtain the samegenomically based delineation by indirect methods provided that descriptions ofindividual genome composition of bacterial genomes are obtained and included inspecies description. The amplified fragment length polymorphism (AFLP) techniquecould provide the necessary data if the nucleotides involved in restriction andamplification are fundamental to the description of genomic divergences. First,in order to verify that AFLP analysis permits a realistic exploration ofbacterial genome composition, we showed the strong correspondence betweenpredicted and experimental AFLP data by using Agrobacterium strain C58 as a modelsystem. Second, we propose a method for determining current genome mispairing andevolutionary genome divergences between pairs of bacteria based upon thearbitrary sampling of genomes with AFLP. The measure of current genome mispairingwas validated by comparison with DNA-DNA relatedness data, which are correlatedto base mispairing. The evolutionary genome divergence is the estimated rate ofnucleotide substitutions that have occurred since strains diverged from a commonancestor. Current genome mispairing and evolutionary genome divergence were usedto compare members of Agrobacterium spp. used as a model of closely relatedgenomic species. A strong and highly significant correlation was found betweencalculated genome mispairing and DNA-DNA relatedness values within genomicspecies. The canonical 70% DNA-DNA hybridization value used to delineate genomicspecies was found to correspond to the range from 13% to 13.6% current genomemispairing. These values correspond to 0.097 and 0.104 nucleotide substitutionsper site, respectively. In addition, experimental data showed that the large Tiand cryptic plasmids of Agrobacterium had little effect upon the estimation ofgenome divergence. The evolutionary genome divergence was used for phylogeneticinferences. Data showed that members of the same genomic species clusteredconsistently as supported by bootstrap resampling. Based upon these results, wepropose that the genomic delineation of bacterial species could be based in thefuture to phylogenetic groups supported by bootstraps, and genome descriptions ofindividual strains obtained by AFLP recorded in accessible data banks, toeventually replace DNA-DNA hybridization studies.