Phylogenetic analyses of 46 species of Iris, representing all subgenera and all sections except Regelia, Brevituba, and Monolepis, utilized matK gene and trnK intron sequence data. Sequence data show that Iris is paraphyletic because Belamcanda chinensis is resolved within the genus. The two largest subgenera, Iris and Limniris, are both resolved as polyphyletic. With the removal of section Hexapogon, subgenus Iris is weakly supported as monophyletic. Relationships within subgenus Limniris are more complex with the subgenus as currently circumscribed representing eight independent origins among the species included in this study. Several potential monophyletic groups are identified including subgenus Scorpiris, series Spuria (subgenus Limniris section Limniris), and a clade of section Limniris species from North America and Asia.