The present study reports on layer chickens with colibacillosis in 2 commercial egg-producing farms (referred to as farm A and farm B, which were managed by the same owner and were about 1 km apart) in the middle region of the Korean peninsula. The 2 flocks were infected at the initiation of egg laying. They were characterized by no previous clinical signs but sudden mortality (2.7-4.0%), with severe lesions of septicemia and fibrinous polyserositis. Escherichia coli was isolated from the lesions of the infected birds. Serotyping tests identified isolates that belonged to somatic groups O1 (12/17), O46 (2/17), O78 (1/17), and O84 (1/17) or that were unidentified (1/17). Thirteen of 17 E. coli isolates (76.4%) obtained from 11 birds in the 2 flocks showed similar pulsed-field gel electrophoresis patterns that were arbitrarily designated as pattern A. The isolates had high frequencies of putative virulence genes including 100% [fimC (type 1 fimbriae), iucD (aerobactin synthesis), and iss (increased serum survival)], 94.1% [cva/cvi (structural genes of colicin V operon) and vat (vacuolating autotransporter toxin)], 88.2% [irp2, iron-repressible protein (yersinia bactin) synthesis, and fyuA, ferric yersinia uptake], and 82.3% [tsh (temperature-sensitive hemagglutinin)]; astA (encoding a heat-stable cytotoxin associated with enteroaggregative E. coli) was not associated with the enteric disorder. These data suggest that all chickens with colibacillosis on farms A and B were likely infected by E. coli strains that are highly pathogenic in avian species.