We report a cladistic analysis of 77 butterfly species of the tribe Melitaeini (Lepidoptera: Nymphalidae) based on mitochondrial DNA gene sequences. We sequenced ca. 536 bp from the 16S ribosomal DNA (rDNA) and a 1422-bp sequence from the cytochrome oxidase I gene. Alignments are critical to statements of homology, especially when aligning rDNA sequences. We aligned the 16S sequences using conventional methods and direct optimization. We found that direct optimization of the sequences produced the best alignments and our preferred phylogenetic hypothesis. Our results suggest that many of the previously proposed genera are paraphyletic and we conclude that there are four monophyletic groups of species in our cladogram: the Euphydryas group, the Phyciodes group, the Chlosyne group, and the Melitaea group. The following genera are found to be paraphyletic: Castilia, Chlosyne, Didymaeformia, Eresia, Melitaea, and Thessalia. In addition, recognition of the monophyletic genera Cinclidia, Mellicta, and Telenassa would render other genera paraphyletic. Our phylogenetic hypothesis indicates that the melitaeines originated in the Nearctic and have colonized the Neotropics three times and the Palaearctic twice.