The introduction of molecular biology methods in clinical microbiology laboratories brings important insights to obtain sensitive and specific diagnoses as fast as possible. These methods are not intended for replacement but for complement of the already applied microbiologic methods. The integrated analyses of all of them is bringing to the most feasible and efficient results. Within the molecular techniques applied, polymerase chain reaction (PCR) has acquired a great diagnostic value, permitting the identification of etiologic agents and the fast and sensitive detection of their virulence and resistance genotypes. Since some years ago, the development of the so called multiplex PCRs has gained deep interest. Those are reactions that get the simultaneous single tube amplification of different target sequences, allowing the simultaneous detection and identification of various genes of interest. In the present article, the most relevant applications of multiplex PCR for clinical microbiology are summarized.