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Background: The Yi people are a sociolinguistic group living in Mile City, which is their typical settlement in southeastern Yunnan, China. Over the long history of using medicinal plants, the Yi people have accumulated and developed a wealth of traditional medicinal knowledge, which has played a vital role in their health care. However, only a few studies have been performed to systematically document the medicinal plants commonly used by the Yi people. This study provides fundamental data for the development and application of ethnomedicine as well as supports the conservation of the traditional medical knowledge of the Yi people. Methods: This study was conducted from May 2020 to August 2022 and involved five townships in Mile. Information regarding medicinal plants was obtained through semistructured interviews, key informant interviews, and participatory observation. The collected voucher specimens were identified using the botanical taxonomy method and deposited in the herbarium. Ethnobotanical data were analyzed using informant consensus factor, relative frequency of citation, and fidelity level. Results: In total, 114 informants distributed in five townships of Mile were interviewed. The Yi people used 267 medicinal plant species belonging to 232 genera and 104 families to treat various diseases. Asteraceae, Lamiaceae, and Fabaceae were the most commonly used plant families by the Yi people. In addition, herbs were most commonly used by the Yi people. Whole plants and roots were the preferred medicinal parts. Decoctions were the most common method of herbal medicine preparation. There are 49 different recorded diseases treated by Yi medicinal plants, and among them, respiratory diseases, rheumatism, traumatic injury, fractures, and digestive system diseases have the largest number of species used. A quantitative analysis demonstrated that plants such as Zingiber officinale, Lycopodium japonicum, Aconitum carmichaelii, Panax notoginseng, Cyathula officinalis, and Leonurus japonicus played crucial roles in disease prevention and treatment. Conclusion: Traditional knowledge of medicinal plants is closely associated with the social culture of the local Yi people. The medicinal plants used for health care in the study area were diverse. Local healers were skilled at using medicinal plants to treat various diseases. Their treatment methods were convenient and unique, exhibiting distinctive regional characteristics. However, the inheritance of their traditional medicinal knowledge and protection of wild medicinal plant resources are facing serious challenges, including the decreasing number of local healers, aging of healers, lack of successors, and excessive harvesting of medicinal plant resources. This ethnobotanical survey provides a useful reference for the sustainable utilization and protection of medicinal plant resources in Mile and the inheritance of traditional medicinal knowledge of the Yi people. [ABSTRACT FROM AUTHOR]