With the increasing indroduction and spread of invasive quarantine pests, accurate diagnosis of pests detected in quarantine sites has become crucial. DNA barcoding, a standardized method that complements morphological analysis for rapid and precise species identification, is actively researched worldwide. In this study, we established a molecular biological identification system for major pests encountered during the import and export of agricultural and forestry products. By analyzing the DNA barcode sequences of pests collected domestically and those detected in quarantine inspections, we compiled genetic information for 1,292 individuals representing 472 species, 108 families across 11 insect orders. Among these, order Lepidoptera had the highest diversity, with 251 species across 27 families. We also secured barcodes for 52 species, 24 families in order Hemiptera, and 70 species, 20 families in order Coleoptera. By constructing a comprehensive biological foundation and database for various pests detected in quarantine sites, we aim to enhance the quarantine system by enabling rapid and accurate identification of invasive pests, thereby blocking early.