Background: During the last decade, coagulase-negative staphylococci (CoNS) have emerged as a major cause of nosocomial infections. They constitute a major component of the normal skin and mucosal microflora, and are particularly responsible for catheter- and medical device-related sepsis. They present unique problems in diagnosis and treatment of infections. Purpose: The present study has been designed to evaluate phenotypic and genotypic characteristics of CoNS among nosocomial isolates. Setting and Design: This study was carried out in a tertiary care hospital. Data from 150 samples collected from 73 hospitalized patients and 15 healthy volunteers between October 2003 and May 2005 were analyzed. Patients and Methods: A total of 100 CoNS strains responsible for sepsis or implant-associated infections and 50 saprophytic strains were studied. Invasive CoNS strains were selected on the basis of different colony morphologies, drug resistance patterns, and biofilm formation. The same criteria were used to select saprophytic isolates. Multiplex PCR was used to explore the ica, mecA, and atlE genes, which might contribute to the pathogenicity of CoNS and the formation of biofilms. Results: Most of the invasive strains that formed the biofilm were resistant to multiple antibiotics, with more than 80% resistant to methicillin. ica and mecA genes were detected significantly in pathogenic strains (chi-square test, P<0.0001) whereas atlE was ubiquitously amplified in all the strains. All those strains which had ica and mecA genes were resistant to multiple antibiotics and were positive for biofilm formation. Conclusion: These genetic markers thus appear to discriminate between potential invasive virulent and saprophytic strains of CoNS.