Abstract Background The northeastern part of the Qinghai-Tibet Plateau (QTP) presents a high number of plateau loach species. As one of the three major groups of fishes distributed on the QTP, plateau loach has high ecological value. However, the taxonomy and systematics of these fish are still controversial, and a large number of new species have been reported. The reason for this phenomenon is that the degree of morphological variation is low, the phylogenetic information provided by morphological and anatomical features used for species identification is relatively poor, and many cryptic species are observed. Based on the high-density sampling points from the biodiversity hotspots surveyed, this study aims to evaluate the biodiversity of plateau loach in the northeastern part of the QTP and reveal the hidden diversity by comparing morphological species with molecular operational taxonomic units (MOTUs). Results After careful identification and comparison of the morphology and DNA barcoding of 1630 specimens, 22 species were identified, with 20 considered valid local species and two identified as new species that had not been previously described. Based on the combination of morphological and molecular methods, a total of 24 native species were found, two of which were cryptic species: Triplophysa robusta sp1 and Triplophysa minxianensis sp1. Fourteen of the 24 species form clusters of barcodes that allow them to be reliably identified. The remaining cases involved 10 closely related species, including rapidly differentiated species and species that seemed to have experienced incomplete lineage sorting or showed introgressions. Conclusions The results highlight the need to combine traditional taxonomies with molecular methods to correctly identify species, especially closely related species, such as the plateau loach. This study provides a basis for protecting the biodiversity of plateau loach.