Aquatic macroinvertebrates serve as vital food sources in freshwater ecosystems. Species identification remains challenging due to morphological similarities, developmental-stage variation, and limited taxonomic expertise. This study assessed the challenges associated with DNA barcoding for species identification of aquatic macroinvertebrates and its application in supporting biological water quality assessment in Kwan Phayao, Thailand. A total of 203 representative samples were analyzed. Morphological identification classified the samples into 13 orders, 7 classes, and 3 phyla, whereas mitochondrial cytochrome c oxidase subunit I (COI) sequences were compared with the reference databases and integrated with phylogenetic analysis and Automatic Barcode Gap Discovery (ABGD), revealing 86 distinct taxa across 37 genera and 29 families. Nonetheless, only 25.62% of the samples were confidently identified at the species level due to limitations in reference databases. Assessment of water quality using physicochemical parameters and biological indices, including the Biological Monitoring Working Party Thai modification (BMWP^Thai) and the Average Score Per Taxon Thai modification (ASPT^Thai), revealed that the water quality of Kwan Phayao was moderate. Accurate identification enabled the detection of taxa with potential public health relevance and economically valuable decapod species, providing preliminary information for the sustainable management and planning of fisheries resources within this ecosystem.

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