Among 75 urosepsis isolates of Escherichia coli, 29 virulence factor (VF) genes were detected by use of a novel polymerase chain reaction (PCR) assay. Compared with probe hybridization, the PCR assay's specificity was 100% and sensitivity 97.1%. fyuA (yersiniabactin: overall prevalence, 93%), traT (serum resistance, 68%), and a pathogenicity-associated island marker (71%) occurred in most strains from both compromised and noncompromised hosts. Present in <20% of strains each were sfaS, focG (F1C fimbriae), afa/dra, bmaE (M fimbriae), gafD (G fimbriae), cnf1, cdtB (cytolethal distending toxin), cvaC (colicin V), and ibeA (invasion of brain endothelium). Different VFs were variously confined to virulence-associated phylogenetic group B2 (as defined by multilocus enzyme electrophoresis); concentrated in group B2, but with spread beyond; or concentrated outside of group B2. These findings provide novel insights into the VFs of extraintestinal pathogenic E. coli and demonstrate the new PCR assay's utility for molecular epidemiological studies.