Prevalence of high-level gentamicin resistance genes aac(6')-Ie-aph(2'')-Ia and aph(2'')-Ie, which encode distinct aminoglycoside-modifying enzymes, was analyzed for a total of 1128 clinical isolates of enterococci obtained in a Japanese hospital during a period between 1997 and 2007. The aac(6')-Ie-aph(2'')-Ia was detected in 40.1%, 12.9%, and 3.6% of Enterococcus faecalis, E. faecium, and other enterococcal species, respectively, and aph(2'')-Ie was detected in 3.3% of E. faecium. During the study period, detection rate of aac(6')-Ie-aph(2'')-Ia in E. faecium increased from 4% (1997-1998) to 28% (2006-2007), whereas generally constant in E. faecalis. By the analysis of IS256-flanking patterns of aac(6')-Ie-aph(2'')-Ia, truncated forms of Tn5281 lacking IS256 at the 3'-end, 5'-end, and both ends of aac(6')-Ie-aph(2'')-Ia were identified in 4.6%, 32.4%, and 34.2% of E. faecalis strains, respectively, while the composite Tn5281-like element with IS256 at both sides was detected in 28.7% of the strains. A truncated form of Tn5281 lacking IS256 at the 5'-end was predominant in other enterococcal species. Among 14 E. faecalis and 10 E. faecium strains harboring aac(6')-Ie-aph(2'')-Ia, 8 and 6 different sequence types (STs) were identified by multilocus sequence typing, respectively. Some E. faecalis STs (ST4, ST16, ST64, and ST223) were found in more than one strain, and ST4 and ST64 were associated with different IS256-flanking patterns. STs of five among six E. faecium strains with aac(6')-Ie-aph(2'')-Ia (ST78, ST203, and ST418) belonged to the clonal complex (CC)17, which is known as globally emerging lineage of vancomycin- or ampicillin-resistant E. faecium clones. E. faecium strains with aph(2'')-Ie were classified into newly assigned STs, ST426, and its single locus variant ST427, which also belonged to CC17. Therefore, it was suggested that E. faecium of CC17 is prone to acquire high-level gentamicin resistance genes, and aph(2'')-Ie is distributed to specific E. faecium clones that are distinct from those having aac(6')-Ie-aph(2'')-Ia.