Gram differentiation through conventional Gram staining is not a reliable method due to high incidence of false positive results. Molecular techniques such as PCR provide a better alternative. A simple, rapid and accurate method has been developed that enables exact differentiation of Gram positive and negative bacteria using a one step PCR assay. The one-step PCR using Gram specific primers resulted in a single 1025bp amplicon in Gram negative bacteria and a 355bp amplicon in addition to 1025bp amplicon in Gram positive bacteria. All tested bacteria were identified correctly through a single PCR reaction and showed complete concordance with biochemical and Gram staining results. This protocol proves to be a rapid, powerful and sensitive tool in determining the Gram status of bacteria. This difference in banding patterns between Gram positive and negative bacteria can be exploited for their clear-cut differentiation.