Background: This study aimed to investigate the occurrence of aminoglycoside resistance and the prevalence of the resistance-modifying enzyme genes, ant(3”)-III, ant(6’)-Ia, aac(6′)-Ie-aph(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.