Fluoroquinolone use in poultry production may select for resistant Escherichia coli that can be transmitted to humans. To define the prevalence and virulence potential of poultry-associated, quinolone-resistant E. coli in the United States, 169 retail chicken products from the Minneapolis-St. Paul area (1999 to 2000) were screened for nalidixic acid (Nal)-resistant E. coli. Sixty-two (37%) products yielded Nal-resistant E. coli. From 55 products that yielded both Nal-resistant and susceptible E. coli, two isolates (one resistant, one susceptible) per sample were further characterized. Twenty-three (21%) of the 110 E. coli isolates (13 resistant, 10 susceptible) satisfied criteria for extraintestinal pathogenic E. coli (ExPEC), i.e., exhibited ≥2 of pap (P fimbriae), sfa/foc (S/F1C fimbriae), afa/dra (Dr binding adhesins), iutA (aerobactin receptor), and kpsMT II (group 2 capsule synthesis). Compared with other isolates, ExPEC isolates more often derived from virulence-associated E. coli phylogenetic groups B2 or D (74% versus 32%; P < 0.001) and exhibited more ExPEC-associated virulence markers (median, 10.0 versus 4.0; P < 0.001). In contrast, the Nal-resistant and -susceptible populations were indistinguishable according to all characteristics analyzed, including pulsed-field gel electrophoresis profiles. These findings indicate that Nal-resistant E. coli is prevalent in retail poultry products and that a substantial minority of such strains represent potential human pathogens. The similarity of the Nal-resistant and -susceptible populations suggests that they derive from the same source population, presumably the avian fecal flora, with Nal-resistance emerging by spontaneous mutation as a result of fluoroquinolone exposure.