In the last decade, along with the problem of nosocomial infections, multidrug-resistant bacteria in community and hospitals have soared. High frequencies of multidrug-resistant bacteria have been grouped under the acronym ESKAPE which capable of ‘escaping’ the biocidal action of antimicrobial agents. The objective of this study is to consider molecular characterization of resistance genes in ESKAPE pathogens. In this study, three hundred and eighty four bacteria were isolated from clinical samples in Loghman-Hakim Hospital, Tehran, Iran. MDR strains reported through disk diffusion method based on CLSI guidline. Molecular characterization of resistance genes were examined by PCR method. The prevalence of MDR-strains of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. were 3(9.09%), 17(23.3%), 11(11.22%), 18(18.36%), 9(18.91%), 4(5.8%), respectively. The most prevalence of resistant genes in ESKAPE bacteria were following as:vanA 40.90%,vanB 22.72% in Enterococcus faecium;mecA24.65%,vanA4.11%, vanB1.37%in Staphylococcus aureus;bla _TEM 28.57,bla_KPC 12.24% in Klebsiella pneumonia;bla_SHV 56.75%,bla_VIM 32.43%,bla _TEM 29.73 %in Acinetobacter baumannii; bla_SHV 44.70%,bla _OXA 55.29%,bla_VEB 74.11%,bla_VIM 62.35%,bla_PER 62.35%,Mex-A 74.11% ,Mex-B 81.17%, Mex-R76.47% , bla_PER 62.35% in Pseudomonas aeruginosa; bla _TEM 39.13%, bla_SHV 33.33% in Enterobacter spp. Knowledge of resistance genes prevalence in ESKAPE pathogens is necessary to prepare feasible data about tracing and treatment of infection related to these microorganisms that may be beneficial to clinicians to select a convenient empirical therapeutic diet in diseases due to ESKAPE pathogens at the bedhead. It is recommended that healthcare-associated, community-acquired, and nosocomial infections to be clearly considered annually.