We conducted a cross-sectional study to determine the prevalence of antimicrobial resistance (AMR) in fecal Escherichia coli isolates from raccoons (Procyon lotor) living in Ontario, Canada. From June to October 2007, we trapped raccoons in three areas: one primarily urban site around Niagara, one primarily rural site north of Guelph, and one at the Toronto Zoo. In addition, we conducted a longitudinal study at the Toronto Zoo site to investigate the temporal dynamics of fecal E. coli and AMR in raccoons. Reduced susceptibility to ≥1 antimicrobial agent was detected in E. coli isolates from 19% of 16 raccoons at the urban site, 17% of 29 raccoons from the rural site, and 42% of 130 samples collected from 59 raccoons at the zoo site. Raccoons from the zoo site were significantly more likely to shed E. coli with reduced susceptibility to ≥1 antimicrobial agent than animals from the rural site (odds ratio [OR], 3.41; 95% confidence interval [CI], 1.17 to 12.09; P = 0.02). Resistance to expanded-spectrum cephalosporins (and the associated bla(CMY-2) gene) was detected in two animals from the zoo site and one animal from the rural site. Serotyping and pulsed-field gel electrophoresis analysis show that raccoons on the zoo grounds harbor a diverse assemblage of E. coli, with rapid bacterial turnover within individuals over time. Our study indicates that raccoons may shed resistant bacteria of public health significance and that raccoons have the potential to disseminate these bacteria throughout their environment.