We determined the prevalence and characteristics of extended-spectrum β-lactamase (ESBL) genes of Enterobacteriaceae in retail chicken meat and humans in the Netherlands. Raw meat samples were obtained, and simultaneous cross-sectional surveys of fecal carriage were performed in 4 hospitals in the same area. Human blood cultures from these hospitals that contained ESBL genes were included. A high prevalence of ESBL genes was found in chicken meat (79.8%). Genetic analysis showed that the predominant ESBL genes in chicken meat and human rectal swab specimens were identical. These genes were also frequently found in human blood culture isolates. Typing results of Escherichia coli strains showed a high degree of similarity with strains from meat and humans. These findings suggest that the abundant presence of ESBL genes in the food chain may have a profound effect on future treatment options for a wide range of infections caused by gram-negative bacteria.