Accurate and rapid diagnostic methods are needed to guide antimicrobial therapy and infection control interventions. Advances in real-time PCR have provided a user-friendly, rapid and reproducible testing platform catalysing an increased use of genetic assays as part of a wider strategy to minimize the development and spread of antimicrobial-resistant bacteria. In this review we outline the principal features of genetic assays in the detection of antimicrobial resistance, their advantages and limitations, and discuss specific applications in the detection of methicillin-resistant Staphylococcus aureus, glycopeptide-resistant enterococci, aminoglycoside resistance in staphylococci and enterococci, broad-spectrum resistance to beta-lactam antibiotics in gram-negative bacteria, as well as genetic elements involved in the assembly and spread of antimicrobial resistance.