Classification of bacterial species into genera has traditionally relied upon variation in phenotypic characteristics. However, these phenotypes often have a multifactorial genetic basis, making unambiguous taxonomic placement of new species difficult. By designing evolutionarily conserved oligonucleotide primers, it is possible to amplify homologous regions of genes in diverse taxa using the polymerase chain reaction and determine their nucleotide sequences. We have constructed a phylogeny of some enteric bacteria, including five species classified as members of the genus Escherichia, based on nucleotide sequence variation at the loci encoding glyceraldehyde-3-phosphate dehydrogenase and outer membrane protein 3A, and compared this genealogy with the relationships inferred by biotyping. The DNA sequences of these genes defined congruent and robust phylogenetic trees indicating that they are an accurate reflection of the evolutionary history of the bacterial species. The five species of Escherichia were found to be distantly related and, contrary to their placement in the same genus, do not form a monophyletic group. These data provide a framework which allows the relationships of additional species of enteric bacteria to be inferred. These procedures have general applicability for analysis of the classification, evolution, and epidemiology of bacterial taxa.