A total of 1921 Escherichia coli isolated from healthy animals (501 from cattle, 832 from pigs, and 588 from chickens) and 237 isolates from diseased pigs were tested to determine the prevalence of apramycin and gentamicin resistance in Korea during 2004-2007. Apramycin/gentamicin resistances observed in healthy cattle, pigs, and chicken were 0.2%/0.6%, 11.2%/13.6%, and 0.5%/18.2%, respectively. Gentamicin/apramycin resistance was much higher in E. coli isolated from diseased pigs (71/237, 30.0%) than in those from healthy pigs (93/832, 11.2%). The aminoglycoside resistance gene content of all apramycin-gentamicin-resistant E. coli isolates (n= 164) was determined by polymerase chain reaction. Of seven different types of aminoglycoside resistance genes tested, five kinds were detected in the 164 isolates: aac(3)-IV, aac(3)-II, aac(3)-III, ant(2'')-I, and armA. All apramycin-resistant E. coli contained the aac(3)-IV gene. About half of the resistant isolates carried only the aac(3)-IV gene and the other half carried other genes in addition to aac(3)-IV. The results of the present study suggest that humans are at risk of gentamicin resistance from apramycin use in animals.