Glycopeptides, such as vancomycin, are frequently the antibiotics of choice for treatment of infections caused by the now common methicillin-resistant Staphylococcus aureus (MRSA). Incidences of vancomycin resistance in S. aureus (VRSA) have been increasing worldwide for the last 5 years. Complex mechanisms producing changes in cell wall content and composition generate the VRSA phenotype, but the genetic basis of these changes has not yet been determined. To facilitate the genetic investigation, entire genome sequences of the archetypal VRSA (Mu50), and vancomycin-susceptible MRSA strains N315, EMRSA 16 and COL were compared. The in silico analysis revealed several loss-of-function mutations in Mu50, affecting important cell wall biosynthesis and intermediary metabolism genes, not previously reported. The new findings provide further evidence for the hypothesis that vancomycin resistance in Mu50 is due to fundamental changes, important to metabolic pathways that impinge on peptidoglycan biosynthesis. These observations will inform targeted experiments aimed at a complete understanding of the mechanism(s) of vancomycin resistance in S. aureus Mu50 and other VRSA strains.