The present study aims to demonstrate the β-lactam resistance phenotypes and genotypes of Escherichia coli isolates from the Fu River in Chengdu, southwestern China. We obtained 108 E. coli isolates from nine sampling sites during May and December 2010. The total bacterial count varied from 79 colony-forming units (CFU)/ml to 14,550 CFU/ml, and coliform group number from 13 to 1,435 MPN/ml. Among the 108 isolates, 0-31.48% were resistant to β-lactams and β-lactam inhibitors, 1.85-7.40% to aminoglycoside, 1-20% to fluoroquinolone, and 50% to trimethoprim- sulfamethoxazole. The total bacterial count and antimicrobial resistance of different sites were significantly correlated (P < 0.05). Among the 34 ampicillin-resistant isolates, polymerase chain reaction (PCR) amplification and DNA sequencing showed that bla (TEM), bla (SHV), and bla (CTX-M) were detected in 85.29% (n = 29), 41.18% (n = 14), and 5.88% (n = 2) of the isolates, respectively, whereas bla (KPC) and bla (GES) were not observed in any of the isolates. Enterobacterial repetitive intergenic consensus-PCR patterns revealed that the 34 ampicillin-resistant E. coli isolates belonged to three distinct groups. Plasmid DNAs from the 14 SHV producer isolates yielded one to five bands of ca. 0.15-40 kb. To our knowledge, the current study is the first to describe the phenotypic and genetic characterizations of β-lactam resistance in E. coli isolates of river water origin from the Fu River, Chengdu, southwestern China. Results of the present study suggest that the river water may be considered as a reservoir for antibiotic resistance genes.