Staphylococcus aureus (including Methicillin-resistant S. aureus) and coagulase negative Staphylococcus spp (including methicillin-resistant coagulase negative) permanently or transiently colonize humans and act as sources of complicated skin and soft tissue infections, bacteraemia and infective endocarditis. They also cause chronic infection of indwelling medical devices. Treatment of these infections has become problematic due to development of methicillin resistance, ability to cause hospital outbreaks and 2-3 days required for their proper identification. Rapid, sensitive detection of methicillin resistant Staphylococcus spp from clinical samples is a necessity. 242 bacterial isolates (162 methicillin-resistant S. aureus and 80 methicillin-resistant coagulase negative S. aureus) from clinical samples was included in the study; detection of mec A, fem A and ica D gene was done by Multiplex-Colony Polymerase Chain Reaction technique. Out of the 242 Staphylococcal strains included in the study, in 192 (79.3%) isolates the presence of mecA gene was detected. Interestingly the presence of fem A gene was detected in all 162 methicillin-resistant S. aureus isolates. However none of the 80 methicillin-resistant coagulase negative S. aureus isolates harbored the fem A gene. The icaD gene Polymerase Chain Reaction revealed that 59.25% of methicillin-resistant S. aureus and 10% of methicillin-resistant coagulase negative S. aureus harbored this gene. Multiplex colony Polymerase Chain Reaction is a comprehensive alternative for rapid sensitive and accurate detection of methicillin resistance and biofilm production in Staphylococcus spp from clinical samples