Objective To investigate the respiratory tract flora and mycoplasma status in Mongolian gerbils from a conventional facility, and provide basic information for the developing microbiologic testing standards of gerbils. Methods Sixty-five gerbils from conventional facility of Capital Medical University were dissected to obtain the tracheal secretion, and then the samples were seeded in blood agar base, cultured in common or 5% CO₂ incubator for 48 h, separately. In order to accurately identify the genus to which the isolated strain belongs, biochemical analysis and PCR sequencing were performed. On the other hand, DNA was isolated from tracheal lixivium, and mycoplasma DNA was amplified by specific primer. The detection rate was calculated. ResultsIn this study, 21 kinds of bacteria and mycoplasma were detected in the tracheas of these mongolian gerbils. The positive rates of different bacterial and mycoplasma infection were varying greatly, between 1.5% (1/65) to 64.6% (42/65), among which, some bacteria were pathogenic to mice and rats. Conclusion This study provides reference information for the development of microbiological standards of Mongolian gerbils.