High-level aminoglycoside resistance (HLAR) limits treatment options for invasive enterococcal infections. We examined the prevalence of HLAR, carriage of genes encoding aminoglycoside-modifying enzymes, and production of β-lactamase using the disk diffusion method, polymerase chain reaction, and a nitrocefin-based test, respectively, in Enterococcus faecalis and Enterococcus faecium isolated from patients at a university hospital in Tokyo in 2010. Of the 100 E. faecalis isolates analyzed, 30 isolates had high-level resistance (HLR) to gentamicin, and 22 isolates had HLR to streptomycin. Of the 40 E. faecium isolates analyzed, 9 isolates had HLR to gentamicin, and 9 isolates had HLR to streptomycin. Of the 39 gentamicin-HLR enterococcal isolates, 24 isolates were non-HLR to streptomycin. All 39 isolates with HLR to gentamicin as well as 19 of 101 without HLR carried aac(6')-Ie-aph(2'')-Ia. Carriage of ant(6')-Ia was confirmed in 25 of 31 streptomycin-HLR isolates. Production of β-lactamase was documented in none of the E. faecalis and E. faecium isolates. Whole-genome sequencing analysis revealed that all but one E. faecalis isolate that carried aac(6')-Ie-aph(2'')-Ia and ant(6')-Ia belonged to sequence type (ST) 4 (n = 8), ST16 (n = 4), or ST179 (n = 9). Nevertheless, most of the pairs of isolates had > 10 single-nucleotide polymorphisms even among the isolates of the same ST.