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The nymphalid butterfly tribe Preponini includes some of the Neotropical region's most spectacular and familiar butterflies, but the taxonomy of the group nevertheless remains unstable. Several recent studies of Nymphalidae phylogeny have suggested that both the tribe itself and several genera might not be monophyletic, but to date taxon sampling has not been sufficiently comprehensive to allow informed revision of the group's systematics. We therefore conducted the first complete species-level phylogenetic study of the tribe to establish a firm higher classification. We used DNA sequence data from three genes, the two mitochondrial genes cytochrome oxidase subunits I and II (COI and COII), and the nuclear gene elongation factor-1α (EF-1α), to reconstruct the phylogeny of the tribe using maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI). We included 48 individuals representing the 22 recognised Preponini species, and an additional 25 out-group taxa to explore taxonomic limits at different levels. Firstly, we found that Anaeomorpha splendida Rothschild never grouped with remaining Preponini, so that maintaining monophyly of the tribe requires the taxon to be excluded, and we thus reinstate the tribe Anaeomorphini stat.rev. Secondly, we investigated generic limits, in particular the relationship of Noreppa Rydon to Archaeoprepona Fruhstorfer, and that of Agrias Doubleday to Prepona Boisduval. The molecular results coupled with previous morphological studies suggest that Noreppasyn.n should be synonymised with Archaeoprepona, and that Agriassyn.n should be synonymised with Prepona. We found Prepona pheridamas (Cramer) to be sister to all other Prepona, and markedly divergent from them in both morphology and DNA sequences, suggesting the possibility that it should be placed in a separate genus. We also found a number of cases of significant DNA sequence divergence and paraphyly or polyphyly within putative species that require further taxonomic attention, including Prepona claudina (Godart) stat.n. and Prepona narcissus (Staudinger) stat.n.,Prepona pylene Hewitson and Prepona deiphile (Godart). Future research should focus on a broader population sampling of widespread, polymorphic Preponini species to thoroughly revise the current species-level taxonomy, thus creating a solid foundation for studies in ecology and conservation.