Avian pathogenic Escherichia coli (APEC) is an important respiratory pathogen of poultry. A variety of virulence-associated genes and serogroups are associated with avian colibacillosis caused by APEC strains. One hundred forty-eight E. coli isolates recovered from diagnosed cases of avian colibacillosis from Guangdong province between 2005 and 2008 were serotyped, and characterized for virulence-associated genes, phylogenetic backgrounds, antibiotic susceptibility, and genetic relatedness. Associations between virulence-associated genes and antimicrobial resistance were further analyzed. Although 148 APEC isolates belonged to 21 different serogroups, 81% were of one of eight serogroups: O65 (27%), O78 (10%), O8 (9%), O120 (9%), O2 (7%), O92 (6%), O108 (5%), and O26 (5%). Polymerase chain reaction analysis showed that the most prevalent gene was traT (90%), followed by iroN (63%), fimH (58%), hlyF (55%), cvaC (54%), and sitA (51%). The APEC strains mainly belonged to groups A (73%) and D (14%). Multiple antimicrobial-resistant phenotypes (greater than or equal to three antimicrobials) were detected in all E. coli isolates, with the majority of isolates displaying resistance to tetracycline (97%), sulfamethoxazole (93%) and fluoroquinolones (87% for ciprofloxacin and 84% for enrofloxacin), chloramphenicol (74%), and florfenicol (66%). All E. coli isolates were further genetically characterized by pulsed-field gel electrophoresis. A total of 125 different pulsed-field gel electrophoresis profiles were obtained, implying that the multiresistant E. coli isolates carrying virulence-associated genes and belonging to multiple serogroups were not derived from a specific clone, but represented a wide variety of chromosomal backgrounds. Statistical analysis showed that several virulence-associated genes were significantly present in APEC isolates susceptibility to multiple antimicrobials. The findings demonstrate that a wide variety of serogroups and potential virulence genes, multiple-resistances, and the clear association of susceptibility and virulence genes have commonly emerged in APEC strains, and these also suggest that antimicrobials should be prudently used to reduce the emergence and spread of resistant strains carrying virulence-associated genes.