To investigate the PCR (polymerase chain reaction, PCR) assay for the detection of Mycoplasma pulmonis in infected rats and try to set up a feasible, quick and sensitive method for the diagnosis. Method 14 samples of the supernatants of throat swab samples and the cultural supernatants of throat swab from infected rats are performed PCR analysis using animal mycoplasms routine primers and M. Pulmonis species-specific primers respectively. The PCR products are confirmed with 2% agarose gel electrophoretic analysis. The M 53 and ATCC19612 standard strain of Mycoplasma pulmonis are used as positive control. Results Using animal mycoplasma routine primers, 8/14 of the supernatants of throat swab samples are positive in the PCR assay whereas 14/14 of the cultural supernatants from throat swab specimen are positive. With M.Pulmonis species-specific primers, the supernatants of throat swab samples are negative in the PCR analysis but 3/14 of the cultural supernatants from throat swab samples are positive. The mycoplasma routine primers can detect the M 53 and ATCC19612 standard strain, on the other hand, M.pulmonis species-specific can only detect M 53 but ATCC19612 strain. Conclusions The PCR assay using mycoplasma routine primers is superior to isolated culture for detecting M. Pilmonis, which could save time and labor. Nevertheless, whether the M.Pilmonis species-specific primers which the foreign researchers suggested could be used to specially detect the M.Pulmonis in the infected rats, need to be further investigated.