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Phylogenetic analysis of mitochondrial DNA (cytochrome c oxidase subunit I; COI) barcode sequences is shown here to be an effective tool to aid in assessing polyphyly in selected genera of the subfamily Eudaminae (Hesperiidae). Results of phylogenetic analyses using:both Bayesian inference and the neighbor-joining (NJ) algorithm were largely congruent and confirm and considerably expand on results from previous studies based on morphology and multilocus genetic data suggesting that the genera Urbanus and Astraptes consist of polyphyletic taxa as currently circumscribed. The genera Narcosius and Ridens, closely-related to Urbanus and Astraptes, emerged as monophyletic, although barcodes were available for only several species from each genus. Amino acid composition of the barcode segment in these and other closely allied genera was also useful in assessing polyphyly. Based on an analysis of barcodes, external morphology and the original description, a reinstated status is proposed for Achalarus jalapus (Plotz, 1881). We suggest that the phylogenetic barcode approach presented here to identify probable misplaced taxa can be used successfully in other taxa of the Eudaminae as well as in other subfamilies of Lepidoptera.