AbstractGlaesserella parasuis,Mycoplasma hyorhinis, andMycoplasma hyosynoviaeare important porcine pathogens responsible for polyserositis, polyarthritis, meningitis, pneumonia, and septicemia causing significant economic losses in the swine industry. A new multiplex quantitative polymerase chain reaction (qPCR) was designed on one hand for the detection ofG. parasuisand the virulence markervtaAto distinguish between highly virulent and non‐virulent strains. On the other hand, fluorescent probes were established for the detection and identification of bothM. hyorhinisandM. hyosynoviaetargeting 16S ribosomal RNA genes. The development of the qPCR was based on reference strains of 15 known serovars ofG. parasuis, as well as on the type strainsM. hyorhinisATCC 17981TandM. hyosynoviaeNCTC 10167T. The new qPCR was further evaluated using 21G. parasuis, 26M. hyorhinis, and 3M. hyosynoviaefield isolates. Moreover, a pilot study including different clinical specimens of 42 diseased pigs was performed. The specificity of the assay was 100% without cross‐reactivity or detection of other bacterial swine pathogens. The sensitivity of the new qPCR was demonstrated to be between 11–180 genome equivalents (GE) of DNA forM. hyosynoviaeandM. hyorhinis, and 140–1200 GE forG. parasuisandvtaA. The cut‐off threshold cycle was found to be at 35. The developed sensitive and specific qPCR assay has the potential to become a useful molecular tool, which could be implemented in veterinary diagnostic laboratories for the detection and identification ofG. parasuis, its virulence markervtaA,M. hyorhinis, andM. hyosynoviae.