Approximately 46% (75/162) or poultry enterococci collected between 1999 and 2000 exhibited high-level resistance to gentamicin (minimum inhibitory concentration [MIC] > or = 500 microg/ml), kanamycin (MIC > or = 500 microg/ml), or streptomycin (MIC > or = 1000 microg/ml). Forty-one percent of the isolates were resistant to kanamycin (n = 67), whereas 23% and 19% were resistant to genramicin (n = 37) and streptomycin (n = 31), respectively. The predominant species identified was Enterococcus faecium (n = 105), followed by Enterococcus faecalis (n = 40) and Enterococcus durans (n = 8). Using polymerase chain reaction, the isolates were examined for the presence of 10 aminoglycoside resistance genes [ant(6)-Ia, ant(9)-Ia, ant(4')-Ia, aph(3')-IIIa, aph(2")-Ib, aph(2")-Ic, aph(2")-Id, aac(6')-Ie-aph(2")-Ia, and aac(6')-Ii]. Five aminoglycoside resistance genes were detected, most frequently aac(6')-Ii and ant(6)-Ia from E. faecium. Seven E. faecalis isolates resistant to gentamicin, kanamycin, or streptomycin were negative for all genes tested, indicating that additional resistance genes may exist. Phylogenetic analysis revealed that the isolates were genetically different with little clonality. These data indicate that enterococci from poultry are diverse and contain potentially unidentified aminoglycoside resistance genes.