Escherichia coli infecting the urinary tract and other extraintestinal sites are often identical in many phenotypic and genotypic characteristics. To discriminate among pathogenic E. coli isolates, chromosomal DNA was digested with restriction endonucleases that recognize eight base pair sequences and the resulting 20-25 fragments were resolved by pulsed field gel electrophoresis (PFGE). Different restriction fragment profiles were detected for isolates obtained from different patients, including isolates of the same evolutionary lineage that were identical in electrophoretic type, serotype, and antibiotic sensitivities. In contrast, isolates obtained from separate sites of infection within the same patient had identical restriction profiles. These results indicate that PFGE can resolve recent evolutionary divergence within E.coli lineages and, consequently, offers a powerful new means for precisely performing molecular epidemiology. In addition, these studies provide genetic evidence that E. coli urinary tract infection within a given patient is typically due to the expansion of a single bacterial clone.