The polymerase chain reaction (PCR) and culture methods were used to detect Mycoplasma pulmonis in nasal, tracheal and oral swab samples of rats derived from 5 mycoplasma-contaminated and 2 mycoplasma-free facilities, and the results of both methods were compared. Thirty-four/54 and 30/54 in nasal samples, 31/54 and 28/54 in tracheal samples, and 12/39 and 30/39 in oral samples were positive in PCR and cultures, respectively. Agreements in the results of both tests were 48/54 (88.9%) in nasal samples and 49/54 (90.7%) in tracheal samples. This indicates that nasal and tracheal samples are useful for detecting M. pulmonis by PCR. The detection number for M. pulmonis from the oral cavity by PCR was remarkably lower than those of nasal and tracheal sites. These results show that the PCR method has significant potential as a rapid and sensitive method for detecting M. pulmonis in clinical samples collected from the nasal cavity and trachea.