DNA barcoding is a powerful tool that provides rapid, accurate, and automatable species identification using standardized genetic region(s), such as for revealing the existence of cryptic species and/or rare species in biodiversity monitoring. DNA barcoding techniques require the development of sets of universal PCR primers for DNA barcoding. We tried to develop universal primer sets, and succeeded in designing not only universal primer sets for DNA barcoding regions of almost all insects, which were designed to include a hypervariable site between highly conserved sites, but also primer sets for longer fragment sequences for registration in a database. We confirmed successful amplification for 14 orders, 43 families, and 68 species with DNA barcoding in the mtDNA 16S rRNA region, and for 13 orders, 42 families, and 66 species with DNA barcoding in the mtDNA 12S rRNA region, including Apterygota and Pterygota (Paleoptera, Polyneoptera, Paraneoptera, and Oligoneoptera). A key feature is that the DNA fragments of the DNA barcoding regions amplified by these primer sets are both short at about 200-bp, and longer fragment sequences will increase the level of data registration in the DNA database. In addition, we evaluated the sensitivity of these newly developed primers using Epeorus aesculus (Heptageniidae), which inhabits a relatively wide range of river systems. The results of this study revealed the existence of a cryptic species or an undescribed species. Such resulting database enhancements will provide opportunities for increasingly accurate assessment of biodiversity and genetic diversity.