ESKAPE group, comprises six bacterial pathogens: Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterococcus faecium, which are major contributors to nosocomial infections due to their opportunistic nature and high levels of antibiotic resistance. this study aimed to assess the prevalence of vancomycin resistance and detect the presence of vanA and vanB genes in S. aureus and Enterococcus isolates collected from hospitalized patients in Mashhad. In this study, S. aureus and Enterococcus isolates were collected from clinical samples of patients during 6 months. All gram-positive isolates of ESKAPE group were identified by standard microbiology and biochemical tests. The sensitivity of the isolates to vancomycin antibiotic was evaluated by disk diffusion and E.test methods. Polymerase chain reaction (PCR) was used to identify vanA and vanB vancomycin resistance genes. The results showed that among 98 Gram-positive ESKAPE group isolates, 21 were Enterococcus species, of which three were resistant to vancomycin. Among these, one isolate carried only the vanA gene, and two isolates carried both vanA and vanB genes. Among 77 S. aureus isolates, 8 were resistant to vancomycin; 3 carried the vanA gene, and 1 carried both vanA and vanB genes. Based on the results, vanA and vanB genes play an important role in multidrug resistance among S. aureus and Enterococcus isolates. Identifying this type of genes, determining the pattern of antibiotic resistance, and also studying their distribution rate is important to determine the epidemiology and control of infections caused by these bacteria.