Many β-lactam antimicrobials, including cephalosporins, have been used in both veterinary and human medicine in the treatment of zoonotic and infectious diseases. Especially, third-generation cephalosporins such as ceftiofur have been approved for systemic use in food-producing animals, which has resulted in the emergence of β-lactamase genes. This study aimed to investigate the occurrence of β-lactamase-producing E. coli isolated from commercial layers and characterized their antimicrobial resistance and virulence genes. Among the 85 cefotaxime (CTX)-resistant E. coli, all isolates showed resistance to at least one antimicrobial, and the rates of resistance to nalidixic acid, cephalothin, ampicillin, and cefazolin were more than 50.0%. In particular, 28 isolates were identified as containing b-lactamase genes. The extended-spectrum β-lactamase (ESBL) and plasmid-mediated AmpC genes blaCTX-M-1, blaCTX-M-14, blaCTX-M-15, and blaCMY-2 were detected in 1, 6, 5, and 4 isolates, respectively. The non-ESBL/pAmpC gene blaTEM-1 was detected in 12 isolates. The distribution of antimicrobial resistance genes in 28 β-lactamase-producing E. coli was as follows: aac(3)-II (64.3%), sul2 (32.1%), tetA (28.6%), sul1 (25.0%), cmlA gene (25.0%), and tetB (14.3%). In total, 6 virulence genes (astA, eaeA, escV, fimH, iucC, and papC) were also identified and the rates in virulence gene were as below: fimH (92.9%), iucC (25.0%), astA (21.4%), papC (10.7%), eaeA (7.1%) and escV (7.1%). Our findings suggest that antimicrobials used in commercial layer must be regulated in Korea, and comprehensive surveillance is necessary to prevent the dissemination of resistant isolates.