Avian pathogenic Escherichia coli (APEC) is the causative agent of colbacillosis in poultry causing economical losses in this industry worldwide. Eighty-four isolates of APEC isolated from diseased chickens in Thailand were examined for their antimicrobial susceptibility. Thirty-four isolates, which were resistance to nalidixic acid or enrofloxacin, were characterized for their mechanisms of fluoroquinolone resistance. In total, isolates demonstrated resistance to tetracycline (84.52%), amoxicillin (70.24%), doxycycline (64.29%), trimethoprim-sulfamethoxazole (51.19%), nalidixic acid (40.48%), enrofloxacin (30.95%), chloramphenicol (28.57%), and gentamicin (9.52%). Twenty-nine isolates contained a mutation in gyrA of a serine at position 83 to a leucine (Ser-83-Leu), and 23 isolates contained a mutation of Asp87 to one one of three amino acids: Asn (n=18), Tyr (n=4) or Gly (n=1). No mutation was found in gyrB. Eighteen isolates contained a mutation in parC of a Ser80 to an isoleucine, and an isolate contained a mutation of Ser87 to an arginine. Furthermore, other point mutations were also found in parC such as Ala-56-Thr (n=1), and Glu-84-Gly (n=1). Six isolates contained a mutation in parE including Ser-458-Ala (n=4), Asp-475-Glu (n=1), and Ile-464-Phe (n=1). In conclusion, the high-level fluoroquinolone resistance in APEC has a correlation with high numbers of mutations in quinolone resistance-determining region (QRDR).