Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of Nosocomial, community acquired infections and neonatal sepsis. The Glycopeptide vancomycin was the drug of choice for treating infections. Aim: Identifying the vancomycin- resistance phenotypically and genotypically among the MRSA isolates from Shibin El-Kom teaching Hospital. Materials and Methods: All samples were collected from Neonatal intensive care unit (NICU) of Shibin El-Kom teaching hospital in Minoufiya, Egypt and identified by conventional methods. S aureus and MRSA were isolated and identified from different clinical samples using conventional methods confirmed by antibiogram of the isolates and mec A gene detection. vancomycin MIC and Vancomycin screening agar were determined following CLSI guidelines. Van A was amplified by PCR using standard primers. Out of the 200 neonates included in this study, 85% were positive growth and 15% were negative growth. Among them, 25% isolates were staphylococci, 42 isolates had nuc gene. Out of 42 S. aureus, 80.95% had mecA gene and 19.05% had not Mec A gene. The VRSA isolates had not van A gene. Conclusions: Vancomycin was still the most effective drug against S. aureus infection. All MRSA in Shibin El-Kom Teaching Hospital had not vanA gene.