Recently, hundreds thousands of CO1 barcodes have been produced in many insect groups however, relatively little is known in Hemiptera. In this study, we newly determine 1173 sequences for 374 species distributed among 190 genera and 33 families collected mostly from North America. Average age of the specimen was 12.2 years and about 70% of the specimens produce barcode over 500 bp among examined 1700 specimens. Con-generic divergency calculated by K2P distance was 9.99 which was 11fold higher than the mean intraspecific variation. A steady increase of genetic variation through increasing taxonomic levels was also observed. We found several taxon groups with deep divergency (over 2.0 K2P distance) which were morphologically identified as a same species. Some mixed clusters with several species were also found and suggested more detailed study for these clusters through both morphological and molecular biological method.