Molecular phylogenetic analyses of hornworts were performed on a nucleotide data set consisting of mitochondrial nad5, chloroplast rbcL and nuclear 18S gene regions. Thirty-seven hornworts representing most of the named generic segregates and morphological diversity in the group were sampled for the three gene analysis while an additional 25 samples were included in an analysis of rbcL alone. Results of these analyses confirm the relationships observed in prior molecular studies but also reveal a number of previously unrecognized yet highly supported clades. On the basis of this and continued morphological and ultrastructural investigations we propose a revised classification of the hornworts. At the highest levels, five well-supported clades are demonstrated and they include: 1) Leiosporoceros, which in most analyses is sister to the remaining hornworts; 2) Anthoceros, Folioceros and Sphaerosporoceros; 3) Notothylas and Phaeoceros; 4) Phymatoceros, a recently described genus that contains two species previously included in Phaeoceros; and 5) the remaining hornwort genera: Megaceros, Dendroceros, Nothoceros and a new genus, Phaeomegaceros. Challenges for continued studies that focus on hornwort systematics include obtaining live specimens worldwide and sorting through the chaotic taxonomy within the group. Morphological studies are hindered by poor preservation of existing collections, and the lack of background data and expertise in hornwort biology. Likewise, molecular sequence data are challenged by the deep divergences and potential effects of extensive RNA editing on phylogenetic reconstruction. Future directions for hornwort systematic studies include accumulating gene sequence data that resolve interspecific relationships and comparative anatomical/ultrastructural work that further elucidates biodiversity in the group. Our ongoing, interdisciplinary studies will culminate in a comprehensive analysis that combines molecular and morphological data for a total evidence approach.