The gut microbiota of bats is vital for their roles in health and the ecosystem, yet studies on hibernating bats in southwest China, particularly in the unique karst landscape of Guizhou, are limited. We captured three hibernating bat species-Pipistrellus (PB), Rhinolophus (RB), and Myotis (MB)-in Liping County, collecting rectal samples for 16S rRNA amplicon sequencing. Data processing involved Trimmomatic, Flash, and Qiime2 for operational taxonomic unit (OTU) standardization and species annotation via the Greengenes database. Differential abundance was analyzed using LEfSe, and diversity metrics were assessed through alpha and beta diversity analyses. The RB group was predominantly composed of