To develop and employ a PCR amplification system, directly from clinical specimens, for the rapid molecular detection of common antimicrobial resistance genes for streptococci, staphylococci and enterococci organisms causing infective endocarditis (IE).Eleven antibiotic resistance genes were targeted by PCR along with four identification-related loci. Blood culture and heart valve material from staphylococcal endocarditis patients were directly examined for methicillin resistance. PCR conditions were optimized for the following antibiotic resistance loci: staphylococci (mecA, aacA-aphD), streptococci (PBP 1A, PBP 2B, gyrB, parE) and enterococci (vanA, vanB, vanC-1, vanC-2, aacA-aphD, aphA3). The presence of methicillin resistance was confirmed in one of the eight IE patients examined.This study presents a PCR amplification system for the detection of antibiotic resistance genes. Detection of such genes may indicate susceptibility of the causal agents of IE to commonly prescribed antimicrobial agents.Rapid detection of antibiotic resistant organisms may reduce the use of inappropriate antibiotic agents or enable the use of the most appropriate combinations of antibiotics, other than those that would normally be prescribed empirically for IE. Such a method may be particularly valuable in cases of culture-negative endocarditis. Detection of antibiotic resistance genes by molecular-based techniques, namely PCR, will allow more directed antibiotic therapy and may also provide opportunities for earlier identification of resistant organisms.