To enhance taxonomic resolution in environmental DNA (eDNA) metabarcoding, we evaluated the utility of the internal transcribed spacer 2 (ITS2) region. We developed ITS2 metabarcoding primers and constructed an ITS2 reference database for Japanese freshwater fishes by sequencing an additional 61 species and compiling existing sequences. We then compared the taxonomic resolution of ITS2 metabarcoding with that of the widely used mitochondrial 12S rRNA (MiFish) marker. Our results demonstrated that ITS2 provided higher resolution, particularly for species in Oncorhynchus and Rhinogobius, which are often difficult to distinguish using MiFish. The improved resolution in Oncorhynchus highlights ITS2's potential for monitoring economically and ecologically important salmonid species. Additionally, ITS2 resolved some closely related species that MiFish could not differentiate, underscoring its value in biodiversity assessments. Our findings suggest that ITS2 can complement MiFish in eDNA-based fish biodiversity monitoring, particularly for taxa where mitochondrial markers have limited resolution. Future studies should further explore the applicability of ITS2 across diverse freshwater ecosystems to optimize its integration into multi-marker metabarcoding approaches.