Abstract There is considerable debate over the contribution of virginiamycin use in animals to quinupristin/dalfopristin (Q/D) resistance in humans. In this study, the prevalence and mechanisms of streptogramin resistance in enterococci from animals and the environment were investigated. From 2000–2004, enterococci from samples were tested for antimicrobial susceptibility. Q/D-resistant isolates (minimum inhibitory concentration ≥4 μg/mL) were subjected to polymerase chain reaction (PCR) using primers for streptogramin resistance genes ( ermB, msrC, vatD and vatE). From the analysis, 1029/6227 (17%) Q/D-resistant non- Enterococcus faecalis enterococci were identified. The majority of Q/D-resistant isolates were Enterococcus hirae ( n = 349; 34%), Enterococcus casseliflavus ( n = 271; 26%) and Enterococcus faecium ( n = 259; 25%). Using PCR, 55.5% ( n = 571) were positive for ermB, 3% ( n = 34) for msrC, 2% ( n = 20) for vatE and 0.3% ( n = 3) for vatD; 39% ( n = 401) were negative for all four genes. The vatD-positive samples comprised two E. faecium from chicken and one E. hirae from swine. The nucleotide sequence of vatD from the three isolates was 100% homologous to published vatD sequences. These data indicate that Q/D resistance among enterococci from animals remains low despite the long history of virginiamycin use. To date, this is the first report of vatD from enterococci in animals in the USA.