The present study was carried out identify Vancomycin A (vanA) gene loci among Methicillin Sensitive Staphylococcus aureus (MSSA) isolates collected from rural and urban tertiary care centers. Identification of species was confirmed by Gram staining, Biochemical and PCR methods. In the study, 16665 samples were analyzed out of which 6538 were collected from rural and 10127 from urban regions. In rural, Methicillin Sensitive Staphylococcus aureus (MSSA) constituted 401 (6.1%) and Methicillin Resistant Staphylococcus aureus (MRSA) 531 (8.1%) samples, where as in urban MSSA constituted 524 (5.2%) and MRSA 771 (7.6%) samples. Vancomycin Sensitive Staphylococcus aureus (VSSA) was isolated by agar dilution method. In rural center was 359 (89.5%), Vancomycin Intermediate Resistant Staphylococcus aureus (VISA) was found in 35 (8.7%) and Vancomycin Resistant Staphylococcus aureus (VRSA) in 7 (1.8%) samples, but in case of urban center VSSA was isolated in 451 (86.1%), VISA in 52 (9.9%) and VRSA in 21 (4.0%) samples. VISA and VRSA strains obtained from agar dilution method were further confirmed by E-Test, Disc diffusion and PCR methods. In E-Test VSSA was present in 2 (4.7%) and 1 (1.4%); VISA in 33 (78.6%) and 51 (69.8%), VRSA in 7 (16.7%) and 21 (28.8%) samples, whereas disc diffusion results revealed VISA in 31 (73.8%) and 66 (90.4%) samples, but PCR revealed VISA in32 (76.2%) and 46 (63.0%), VRSA in 7 (16.7%) and 21 (28.8%) samples collected from rural and urban centers. Two strains of vanA locus subjected to PCR amplification, Sanger sequencing and sequencing data were analyzed using BLAST NR database of NCBI genbank and results were compatible with Vancomycin-Resistant Enterococci (VRE) and Vancomycin-Resistant Staphylococci aureus (VRSA). Finally, vanA gene loci could be present in any Gram positive bacteria, which was neither to Methicillin sensitive nor resistant. © Triveni Enterprises.