We conducted quarantine insect species diversity monitoring using DNA barcoding with 517 lepidopteran samples that were obtained from quarantine inspections of foreign vessels entering Korea. The DNA barcode of each sample was treated as a molecular operational taxonomic unit (MOTU). For species delimitation and species identification of the analyzed samples, we applied a 2% cutoff rule and then identified with using BLAST of NCBI GenBank and BOLD System ver. 3.0. Consequently, 517 analyzed samples were delimited as 214 putative species across 20 families. Of these 214 putative species 145 (368 samples) were considered taxonomically identified if the closest BLAST match was no more than 2% different. Therefore the number of samples that were identified to the species level was relatively low, at approximately 71%. 115 of the 145 species were known in Korea. Of the 30 species that were not known in Korea, three, i.e., Noctua pronuba (Noctuidae), Orthosia hibisci (Noctuidae), and Pieris brassicae (Pieridae), were checked as ‘Regulated pests’ in Korea. We suggest that the three regulated pest species could be prevented from being introduced to Korea if monitoring of the vessels that pass the navigation route that contains these three species is performed consistently. Therefore, we suggest that the monitoring of quarantined insect pest enables the prevention of the introduction of alien species.