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ABSTRACT Accurate identification of species is fundamental to every biological research. While morphological identification is a time-consuming and skilled technique, straightforward molecular techniques require the availability of a database of previously sequenced and identified specimens. For most countries of South America, species of noctuids with available sequences are scarce, mostly restricted to species of economic importance, making molecular identification untenable. Here we sequenced the mitochondrial DNA ‘barcode’ region of the cytochrome c oxidase subunit I gene of 34 specimens pertaining to 26 species of South American noctuids, 20 of these for the first time. For all species, genetic distances were higher for interspecific than for intraspecific relations, supporting the utility of DNA ‘barcodes’ to identify species. Larger intraspecific divergences occurred in species of Agrotis and Anicla, and interspecific divergences lower than 2% occurred in about one fifth of the species, all in species of Agrotis, Feltia, and Anicla. These results will allow identifying these species using DNA ‘barcodes’, either for pest management or general biological studies. Furthermore, we carried out phylogenetic analyses with those sequences and 158 other sequences of 85 species of noctuids mined from GenBank. These analyses, in every case, grouped species of the same genus suggesting that the DNA ‘barcodes’ region alone can be useful for lower level phylogeny in this group, recovering as monophyletic groups such as Copitarsia, Agrotis and Austrandesiita. Conversely, groups such as Mythimna (Mythimna) and the subgenera of Feltia were not recovered as monophyletic, suggesting the need for further taxonomic studies in these groups.