The layer operation system has a pyramidal structure in which the grandparent stock (GPS) is at the top, followed by the parent stock (PS), and commercial chickens (CC) that produce eggs at the bottom of the pyramid. Unfortunately, this vertical integration of the layer industry allows the transfer of Escherichia coli(E. coli) to the next step. The objective of this study was to genetically characterize and investigate the prevalence of third-generation cephalosporin-resistant E. coli at all levels of the layer operation system in Korea. A total of 561 E. coli samples were tested in this study, and antimicrobial resistance to third-generation cephalosporin, cefotaxime, and ceftazidime gradually increased from GPS to CC. Fifty-one (9.1%) isolates produced -lactamase genes: GPS harbored bla(TEM-1) (1 isolate) or bla(CMY-2) (2 isolates) genes; PS harbored bla(TEM-1) (7 isolates) or bla(CMY-2) (6 isolates) genes; CC harbored bla(CTX-M-1) (1 isolate), bla(CTX-M-14) (1 isolate), bla(TEM-1) (13 isolates), bla(CTX-M-14+TEM-1) (7 isolates), or bla(CMY-2) (4 isolates) genes; and eggs harbored bla(CTX-M-1) (2 isolates), bla(CTX-M-14) (1 isolate), bla(TEM-1) (3 isolates), or bla(CMY-2) (3 isolates) genes. Pulsed-field gel electrophoresis (PFGE) analysis was performed on the 51 -lactamase-producing E. coli isolates. In PFGE, E. coli included 7 PFGE patterns showing the same production stage and exhibited both -lactamase genes and the antimicrobial resistance pattern. Our findings suggest that there is a critical need for comprehensive surveillance of third-generation cephalosporin-resistant E. coli at all levels of the layer-production pyramid and provide important considerations for the control of infection in large-scale layer operations in Korea.