Abstract: Actinobacillus pleuropneumoniae (App), Haemophilus parasuis (Hps), Bordetella bronchiseptica (Bb), Toxigenic Pasteurella multocida (T+Pm) and Mycoplasma hyopneumoniae (Mhp) are five kinds of the most common pathogenic bacteria which can cause a series of respiratory tract diseases in swine. Five pairs of specific oligonucleotide primers were designed according to the DNA sequences of App apxIV, Hps 16S rRNA, Bb fla, toxigenic T+Pm toxA and Mhp ldh reported in NCBI website. A multiple PCR detection method was established according to improved systems and programmes on the basis of five single PCR methods. No cross reaction was detected by the multiplex PCR method between these five bacteria templates and other common bacteria templates. The sensitivity of multiple PCR was lower than 160 pg genome template. Using this multiple PCR method, bacteria could be detected from lung tissues of challenged mice with App, Hps, Bb and T+Pm respectively after being cultured for 8 h in Tryptic Soy Broth (TSB) supplemented with 5% calf serum and NAD+ factor. Simultaneous detection of pathogens in 269 clinical samples from the central area of China revealed the universal existence of App, Hps, Bb, T+Pm and Mhp. In conclusion, the multiple PCR assay showed excellent specificity, sensitivity and reduplication and could detect five pathogenic bacteria of respiratory tract in swine. We expected that this multiple PCR assay could be useful extensively for epidemiological survey and clinical differential diagnosis of five common respiratory diseases in swine.