The exotic Sirex woodwasp (Sirex noctilio), recently introduced into native pine forests of the northeastern US and Canada, will likely become a major pest of pines and possibly other conifers in North America. This eurasian siricid woodwasp causes damage by the injection of a phytotoxic mucus and inoculum of a damaging exotic wood decay fungus (Amylostereum areolatum) into living pine trees at oviposition. Larvae feed on pine wood decayed by the fungus and on the fungus itself, producing large galleries in the wood. S. noctilio larvae are morphologically indistinguishable from endemic North American siricid larvae. The aim of this study was to develop a diagnostic molecular method for identifying native and nonnative siricoid woodwasps in North America. A modified DNA barcode method was tested as a means for identifying and distinguishing S. noctilio larvae from native woodwasp larvae in wood based on PCR amplification and sequencing of a 680-basepair region of the mitochondrial gene cytochrome c oxidase I (CO1). Comparison of DNA homology within the barcode region between S. noctilio and 22 native woodwasps showed numerous locations in this gene where differences in base substitutions were detected, allowing effective identification and discrimination from all North American woodwasps tested. These methods also are useful for identifying nonnative woodwasp larvae detected or encountered in wood during wood-importation inspections at US and Canada ports of entry, in forest exotic insect-pest surveys and for measuring taxonomic relatedness between woodwasp species. The woodwasp-specific, CO1-based DNA barcode method developed here has proven to be a reliable and efficient new tool useful for the rapid and accurate identification of siricoid woodwasp larvae and adults. The significance and possible limitations of DNA barcode methods for biosecurity applications are discussed.