Properly identified specimen databases that include DNA sequences are mandatory for studies on species identification by DNA barcodes, metabarcoding of biota, and environmental DNA (eDNA) analyses. In addition, high-resolution digital images of well-characterized specimens are useful for morphology-based taxonomy studies. We have isolated the genomic DNA of physical collections (specimens) of Japanese aquatic macroinvertebrates that were stored at the authors’ laboratory and determined partial coding regions for 18S rRNA, histone H3, and mitochondrial cytochrome c oxidase subunit I (COI) genes. Through this work, digital data (photographs and DNA sequences) on aquatic macroinvertebrates inhabiting Japan, along with their collection data, have been made available on the J-amir website (http://www.b.s.osakafu-u.ac.jp/~mkato/J-amir_home.htm). As of March 2020, our collections included 75 taxa of Ephemeroptera (out of 152 species known to inhabit Japan), 30 taxa of Odonata (out of 211 species), 11 taxa of Plecoptera (out of 203 species), and 33 taxa of Trichoptera (out of 484 species), in addition to data of other orders. The website will be continuously updated with new data. Our collection offers a reference for species identification by DNA barcodes, quick image matching of aquatic macroinvertebrates, and evaluating the molecular phylogeny of aquatic insects.