Although many strain typing methods exist for pathogenic Escherichia coli, most have drawbacks in terms of resolving power, interpretability, or scalability. For this reason, multilocus sequence typing (MLST) is an appealing alternative. However, its applicability to different pathogens in specific epidemiologic contexts is not well understood. Here, we applied a previously established MLST method based on housekeeping genes to a well-characterized collection of uropathogenic E. coli isolates to compare the discriminatory ability of this procedure with that of enterobacterial repeat intergenic consensus (ERIC2) PCR, serogrouping, and pulsed-field gel electrophoresis (PFGE). Among 45 E. coli isolates studied, 17 different multilocus sequence types (ST) were identified. One MLST group (designated ST69 complex) was comprised of 22 isolates, all belonging to uropathogenic and bacteremic E. coli strains previously defined as clonal group A (CgA) by ERIC2 PCR. The ST69 strains contained five different serogroups and 14 PFGE types. ERIC2 PCR CgA strains belonging to different MLST groups were also identified. Interestingly, one cow E. coli isolate, previously shown by PFGE to be closely related to a human uropathogenic CgA strain, was found to cluster with the ST69 strains. All of the other animal and environmental CgA isolates had different MLST profiles. The discriminatory power of this MLST method based on housekeeping genes appears to be higher than that of ERIC2 PCR but lower than that of PFGE for epidemiologic study of uropathogenic E. coli.