Abstract: This study reports DNA barcodes for more than 1300 Lepidoptera species from the eastern half of North America, establishing that 99.5% of these species possess diagnostic barcode sequences. Intraspecific divergences averaged just 0.43% among this assemblage, but most values were lower. The mean was elevated by deep barcode divergences (>2%) in nearly 5% of the species, often involving the sympatric occurrence of two barcode clusters. A few of these cases have been analyzed in detail, revealing species overlooked by the current taxonomic system. This study also provided a large-scale test of the extent of regional divergence in barcode sequences, indicating that geographic differentiation is small, even when comparisons involve populations as much as 2800 km apart. The present results affirm that a highly effective system for the identification of Lepidoptera can be built with few records per species because of the limited intra-specific variation. As most terrestrial and marine taxa possess patterns of population structure similar to those in Lepidoptera, an effective DNA-based identification system can be developed with modest effort.