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The adoption of DNA-based assessments for biodiversity monitoring has been on the rise. However, the effectiveness of DNA-based taxonomic assignments heavily relies on the availability and reliability of DNA barcode libraries. There is growing demand for a comprehensive understanding of aquatic biodiversity and the critical role of Chironomidae, specifically Stenochironomus in freshwater ecosystems. Therefore, our objective is to develop a reference barcode library for Stenochironomus in China. From 2016 to 2021, we collected Stenochironomus specimens in diverse Chinese landscapes using malaise traps, light traps, and sweep nets. These specimens were carefully preserved for DNA extraction and barcode sequencing. Our analysis unveiled 36 unique operational taxonomic units from 180 COI barcode sequences through a Neighbor-Joining tree and Automatic Barcode Gap Discovery program, highlighting a significant diversity within the Stenochironomus species. The findings emphasize the constraints of conventional morphological identification methods, especially for species with ambiguous morphologies. It also underscores the effectiveness of DNA barcoding in revealing hidden species diversity, known as cryptic species. Consequently, this study advocates for an integrated taxonomic approach, combining morphological and molecular data, to refine species identification and conservation strategies.