Objective The aim of this study is to establish a multiplex polymerase chain raction (PCR) to identify of four kinds of laboratory animal pathogens:Pasteurella multocida, Bordetella bronchiseptica, Mycoplasma pneumoniae and Klebsiella pneumoniae. Methods Specific primers were designed based on GenBank data. The multiplex PCR system was established through optimization of multiple PCR and detection of its specificity and sensitivity. This technique was used to test artificially infected samples and tracheal secretions of experimental animals (rat, mouse, guinea pig, rabbit, hamster), and comparing the detection results by this method and traditional detection test. Results Target bands of Pasteurella multocida (356 bp), Bordetella bronchiseptica (237 bp), Mycoplasma pneumoniae (266 bp), and Klebsiella pneumoniae (142 bp) were obtained, with a detection sensitivity of Klebsiella pneumoniae of 10 pg, and that of Pasteurella multocida, Bordetella bronchiseptica and Mycoplasma pneumoniae of 1 pg by this newly developed multiplex PCR assay. No target bands were observed from the non-specific pathogens of artificially infected samples. The tracheal secretions taken from 45 experimental animals (mice and rabbits) were tested with this new PCR assay, among which 15 cases of Klebsiella pneumonia and 9 cases of Pasteurella multocida were detected as positive, while all the results of traditional method and serological test were negative. Conclusions A simple, rapid, specific and highly sensitive multiplex PCR system has been successfully established.It is valuable for detection of Pasteurella multocida, Bordetella bronchiseptica, Mycoplasma pneumoniae, and Klebsiella pneumoniae in laboratory animals.