Aquatic Coleoptera and Hemiptera insects primarily inhabit lentic waters, many of which are at risk of extinction due to development, agriculture, and invasive alien species. Environmental DNA (eDNA) analysis has recently emerged as a powerful tool for conducting comprehensive distribution surveys. The cytochrome c oxidase subunit I (COI) universal primers are conventionally used for DNA barcoding by non-specific amplification and frequent amplification failures. Primers in the mitochondrial 16S region that alleviate these issues have been developed and are considered helpful for eDNA analysis. Therefore, it is necessary to accumulate reference sequences of the 16S region in aquatic Coleoptera and Hemiptera insects. However, molecular identification at the genus or species level remains challenging, as only a few of these insect groups in Japan have registered both COI and 16S reference DNA sequences. Therefore, we constructed a comprehensive dataset of the 16S region of mitochondrial DNA for these insects distributed in Japan. As a result of this study, we were able to obtain partial sequences of the 16S region from 140 taxa (35.5% of Japanese aquatic Coleoptera species or subspecies) and 58 taxa (45.3% of Japanese aquatic Hemiptera species or subspecies). These voucher specimens were deposited in four research institutions. The DNA sequence datasets are expected to significantly contribute as an essential database for eDNA analysis and other DNA metabarcoding studies.