Heterogeneous vancomycin intermediate Staphylococcus aureus (hVISA) strains are increasingly reported, and their association with vancomycin treatment failure is a well-known problem worldwide. A total of 500 clinical isolates of methicillin-resistant S. aureus were screened for hVISA by four different methods from May 2011 to May 2014. The number of strains screened for hVISA from May to April in 2011-2012, 2012-2013, and 2013-2014 were 198, 123, and 179, respectively. hVISA strains were studied by transmission electron microscopy (TEM) for cell wall thickness and also for their ability to form biofilm on a polystyrene microtiter plate. hVISA strains detected by four different methods-brain heart infusion agar with vancomycin with 4 mg/L/gradient plate/macro E-test/and glycopeptide resistance detection (GRD) E test-were as follows: 11.6%/10%/9%, and 9.5% in 2011-2012, 12.1%/9.7%/8.9%, and 10.5% in 2012-2013, and 13.9%/11.7%/11.1%, and 12.8% in 2013-2014, respectively. Population analysis profile-area under curve analysis confirmed hVISA in 4.5% (9/198), 6.5% (8/123), and 6.7% (12/179) in respective years; 24% (7/29) of hVISA isolates were nonsusceptible to daptomycin. TEM showed a significant increase in cell wall thickness of hVISA isolates (p<0.001) with a distinct reduction in their biofilm formation ability.