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Faeces Vespertilionis is a commonly used fecal traditional Chinese medicine. Traditionally, it is identified relying only on morphological characters. This poses a serious challenge to the composition analysis accuracy of this complex biological mixture. Thus, for quality control purposes, an accurate and effective method should be provided for taxonomic identification of Faeces Vespertilionis. In this study, 26 samples of Faeces Vespertilionis from ten provinces in China were tested using DNA metabarcoding. Seven operational taxonomic units (OTUs) were detected as belonging to bats. Among them, Hipposideros armiger (Hodgson, 1835) and Rhinolophus ferrumequinum (Schober and Grimmberger, 1997) were the main host sources of Faeces Vespertilionis samples, with average relative abundances of 59.3% and 24.1%, respectively. Biodiversity analysis showed that Diptera and Lepidoptera were the most frequently consumed insects. At the species level, 19 taxa were clearly identified. Overall, our study used DNA metabarcoding to analyze the biological composition of Faeces Vespertilionis, which provides a new idea for the quality control of this special traditional Chinese medicine. [ABSTRACT FROM AUTHOR]