Abstract: Objective To investigate the effect of repeated freeze-thaw on the culture and isolation of Mycoplasma pneumoniae (M. pneumoniae) from throat swab specimen and improve the isolation of M. pneumoniae from clinical specimen. Methods A total of 150 throat swabs were taken from the pneumonia patients in hospitals in Beijing. After culture the viable organism in these specimens were detected by color changing unit (CCU) assay, the positive culture rate and/or viable organism amount were analyzed for repeated freeze-thaw specimens (1 to 4 times). Results There were about 0-103CCU of M. pneumoniae in each 200 l clinical swab specimen. The specimens with M. pneumoniae less than 10 CCU accounted for 40% of the total positive specimens and 25% of such specimens turned to be negative after 1 time freeze-thaw. After three times freeze-thaw, specimens with more than 10 CCU of M. pneumoniae had no significant change in CCU count, but which needed about 14 hours for prolonged incubation. Conclusion Due to the organisms sensitivity to repeated freeze-thaw, the freeze-thaw of clinical specimen should be avoided before culture.