To investigate the epidemiologic aspects of colibacillosis in broiler chickens, 83 Escherichia coli isolates obtained from the pericarditis and perihepatitis lesions in broiler chickens from 4 commercial farms, 5 isolates recovered from 5 samples of yolk sac contents that were pooled from 25 emaciated chicks, and 4 fecal isolates obtained from a hatchery that supplied chicks to the 4 commercial farms mentioned above were genetically and bacteriologically characterized. Using pulsed-field gel electrophoresis (PFGE), a total of 92 isolates were classified into 33 pulsotypes. Identical pulsotypes were observed in isolates obtained from hatchery samples and the affected broiler chickens on multiple farms at various sampling times. Seventeen representative isolates with no common origin belonging to 6 pulsotypes and an additional 27 isolates with the other pulsotypes were used for further experiments. Isolates with identical pulsotypes exhibited common traits for virulence-associated genes, lipopolysaccharide core types, and phylogenetic groups. Nine of the isolates were serologically typed as O125 with various types of H antigens and 3 were typed as O25:H4. In the 27 isolates resistant to ceftiofur (CTF), which is a third generation cephalosporin, the blaCTX-M-2, blaCMY-2, blaCTX-M-14, blaCTX-M-65 genes were found in 15, 8, 3, and 1 isolate(s), respectively, and another isolate resistant to CTF had both the blaCTX-M-2 and the blaCMY-2 genes. In the 16 isolates with the blaCTX-M-2 gene, the chromosomal location of the gene was identified in 12 isolates. The plasmid-mediated quinolone resistance genes, oqxAB and aac(6')-Ib-cr, were found in 2 and 3 isolates, respectively. Conjugation experiments revealed that the blaCTX-M-2 (4 isolates), blaCTX-M-14 (3 isolates), blaSHV-12 (1 isolate), and oqxAB (2 isolates) genes were transferred. Our data suggest that E. coli strains with identical pulsotypes had been caused the incidences of colibacillosis and that the antimicrobial resistance genes on conjugative plasmids and those integrated into the chromosome may be spread among avian pathogenic E. coli strains in multiple farms.