Sisyrinchium (Iridaceae) is a New World genus of approximately 140 species. Seventy-five accessions from sixty species representing the geographical and morphological diversity present within the genus were included in this study. Maximum likelihood and Bayesian inference analyses utilized ITS and matK/trnK datasets, analyzed separately and combined. Based on this study the genus Sisyrinchium is monophyletic and is sister to Olsynium. The currently accepted subgenera, Echthronema and Sisyrinchium, are not monophyletic. Instead, all analyses revealed eight major clades, which correspond to named sections or are given informal clade names. Relationships among the eight major clades are resolved except for early diverging clades where topologies for the two DNA-region trees did not agree. Additionally, several widespread species are not monophyletic based on data from these two molecular markers.