Enterococci isolated from a bison population on a native tall-grass prairie preserve in Kansas were characterized and compared to enterococci isolated from pastured cattle. The species diversity was dominated by Enterococcus casseliflavus in bison (62.4%), while Enterococcus hirae was the most common isolate from cattle (39.7%). Enterococcus faecalis was the second most common species isolated from bison (16%). In cattle, E. faecalis and Enterococcus faecium were isolated at lower percentages (3.2% and 1.6%, respectively). No resistance to ampicillin, chloramphenicol, gentamicin, or high levels of vancomycin was detected from either source. Tetracycline and erythromycin resistance phenotypes, encoded by tetO and ermB, respectively, were common in cattle isolates (42.9% and 12.7%, respectively). A significant percentage of bison isolates (8% and 4%, respectively) were also resistant to these two antibiotics. The tetracycline resistance genes from both bison and cattle isolates resided on mobile genetic elements and showed a transfer frequency of 10−6 per donor, whereas erythromycin resistance was not transferable. Resistance to ciprofloxacin was found to be higher in enterococci from bison (14.4%) than in enterococci isolated from cattle (9.5%). The bison population can serve as a sentinel population for studying the spread and origin of antibiotic resistance.