We evaluated the molecular mechanism for resistance of 360 enterococci for which the gentamicin MICs were ≥128 μg/ml. The aac(6′)-Ie-aph(2″)-Ia, aph(2″)-Ic, and aph(2″)-Id genes were identified by PCR in isolates from animals, food, and humans. The aph (2″)-Ib gene was not identified in any of the isolates. Two Enterococcus faecalis isolates (MICs > 1,024 μg/ml) from animals failed to generate a PCR product for any of the genes tested and likely contain a new unidentified aminoglycoside resistance gene. Pulsed-field gel electrophoresis (PFGE) analysis showed a diversity of strains. However, 1 human and 18 pork E. faecalis isolates from Michigan with the aac(6′)-Ie-aph(2″)-Ia gene had related PFGE patterns and 2 E. faecalis isolates from Oregon (1 human and 1 grocery store chicken isolate) had indistinguishable PFGE patterns. We found that when a gentamicin-resistant gene was present in resistant enterococci from animals, that gene was also present in enterococci isolated from food products of the same animal species. Although these data indicate much diversity among gentamicin-resistant enterococci, the data also suggest similarities in gentamicin resistance among enterococci isolated from humans, retail food, and farm animals from geographically diverse areas and provide evidence of the spread of gentamicin-resistant enterococci from animals to humans through the food supply.