In total, 83 avian pathogenic Escherichia coli (APEC) isolates from avian colibacillosis during a period from 2001 to 2006 in Japan were investigated for serogroups, typical virulence factors, antimicrobial susceptibility, and genetic relatedness. The most common serogroup was O78 (30.1%); 80.7% of isolates harbored the iss gene and 55.4% of isolates harbored the tsh gene. Antimicrobial resistance of the isolates was found for ampicillin (77.1%), oxytetracycline (75.9%), kanamycin (36.1%), fradiomycin (33.7%), trimethoprim (25.3%), enrofloxacin (21.7%), and florfenicol (6.0%). Although multiple antimicrobial-resistant phenotypes (three or more antimicrobials) accounted for 54.2% of isolates, no isolate exhibited resistance to all agents tested. The fluoroquinolone-resistant isolates had point mutations in GyrA (Ser83 --> Leu, Asp87 --> Asn) and ParC (Ser80 --> Ile, Glu84 --> Gly). Of 18 enrofloxacin-resistant E. coli isolates, nine isolates belonged to serotype O78. In PFGE analysis, eight of the nine enrofloxacin-resistant O78 isolates were classified into an identical cluster. This suggests that a specific genotype of fluoroquinolone-resistant O78 APEC may be widely distributed in Japan.