BACKGROUND: The China Space Station (CSS) modules feature many areas that are difficult to clean and thus susceptible to microbial outbreaks. A new sampling method utilizing an equivalent material sheet was applied to characterize the diversity of microbes that accumulated in inaccessible areas in orbit on the CSS. Equivalent material sheet is a membrane made of the same material as the wall of the module. RESULTS: Fifty samples were collected from interior surfaces (work, sleeping, and sanitary areas) of the Tianhe core module and the Wentian and Mengtian experimental modules, covering three flights by the Shenzhou (SZ)-12 to SZ-14 astronaut crews from 2021 to 2022. The numbers of culturable bacteria and fungi that accumulated during the on-orbit periods of each flight ranged from 0 to 2.83 × 10 CONCLUSION: Future efforts at microbial prevention and control on orbit should emphasize the human and plant origins of microbes. Information on the microbial diversity in the condensate zone could be useful to guide the development of new strategies to prevent and control microbes during space flight. Video Abstract.