Aims: Escherichia coli have been targeted for studying antimicrobial resistance in companion animals because of opportunistic infections and as a surrogate for resistance patterns in zoonotic organisms. The aim of our study is to examine antimicrobial resistance in E. coli isolated from various anatomical sites on healthy dogs and cats and identify genetic relatedness. Methods and Results: From May to August, 2007, healthy companion animals (155 dogs and 121 cats) from three veterinary clinics in the Athens, GA, USA, were sampled. Escherichia coli was isolated from swabs of nasal, oral, rectal, abdomen and hindquarter areas. Antimicrobial susceptibility testing against 16 antimicrobials was performed using broth microdilution with the Sensititre (TM) system. Clonal types were determined by a standardized pulsed-field gel electrophoresis protocol. Although rectal swabs yielded the most E. coli (165/317; 52%) from dogs and cats, the organism was distributed evenly among the other body sites sampled. Escherichia coli isolates from both dogs and cats exhibited resistance to all antimicrobials tested with the exception of amikacin, cephalothin and kanamycin. Resistance to ampicillin was the most prevalent resistance phenotype detected (dogs, 33/199; 17%; and cats, 27/118; 23%). Among the resistant isolates, 21 resistance patterns were observed, where 18 patterns represented multidrug resistance (MDR; resistance 2 antimicrobial classes). Also among the resistant isolates, 33 unique clonal types were detected, where each clonal type contained isolates from various sampling sites. Similar resistance phenotypes were exhibited among clonal types, and three clonal types were from both dogs and cats. Conclusions: Healthy companion animals can harbour antimicrobial-resistant E. coli on body sites that routinely come in contact with human handlers. Significance and Impact of the Study: This study is the first report that demonstrates a diverse antimicrobial-resistant E. coli population distributed over various sites of a companion animal's body, thereby suggesting potential transfer of resistant microflora to human hosts during contact.