Background: This study aimed to investigate the occurrence of aminoglycoside resistance and the prevalence of the resistancemodifying enzyme genes, ant(3” )-III, ant(6’ )-Ia, aac(6′ )-Ieaph( 2” )-Ia, and aph(2’ )-Id, in Enterococcus strains isolated in Kermanshah Province, west of Iran. Methods: In this cross-sectional study, 108 enterococcal isolates from urine, wound, blood, and cerebrospinal fluid samples were collected. The Enterococcus species were recognized by standard phenotypic/biochemical tests. The antimicrobial resistance forms were detected using a disc diffusion method. Polymerase chain reaction was designed to identify aminoglycoside resistance genes, including ant(3” )-III, ant(6’ )-Ia, aac(6′ )-Ie-aph(2” )-Ia, and aph(2’ )-Id. Results: Totally, 108 strains with a final diagnosis of Enterococcus were extracted from 84 (77. 8%) urine, 14 (13%) wound, 6 (5. 6%) blood, and 4 (3. 7%) cerebrospinal fluid samples. Among the 108 Enterococcus specimens, 94 (87%) cases were Enterococcus faecalis and 14 (13%) were Enterococcus faecium. The highest frequency of resistance was observed for erythromycin (88. 9%), while the lowest was found for streptomycin (44. 4%). The frequency of high-level gentamicin resistance was 42. 2%. Among the identified specimens, 42. 6% contained the aac(6′ )-Ie-aph(2” )-I gene, 20. 4% contained the ant(6’ )-Ia gene, and 15. 7% contained the ant(3” )-III gene. A significant correlation was found between phenotypic gentamicin resistance and the presence of the aminoglycoside resistance genes (P<0. 05). Conclusion: This study showed the high resistance of Enterococcus strains isolated from hospital samples. Compared with the previous studies, the strains isolated in our study showed a higher percentage of resistance to aminoglycosides.