© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Taxus is a genus of trees and shrubs with high value in horticulture and medicine as a source of the anticancer drug paclitaxel. The taxonomy of the group is complex due to the lack of diagnostic morphological characters and the high degree of similarity among species. Taxus has a wide global geographic distribution and some taxonomists recognize only a single species with geographically defined subgroups, whereas others have described several species. To address these differences in taxonomic circumscription, phylogenetic analyses were conducted on DNA sequences using Maximum Likelihood, Bayesian Inference and TCS haplotype networks on single and combined gene regions obtained for the nuclear ribosomal ITS region and the plastid trnL intron and trnL-F intergenic spacer. Evidence is presented for the sister group status of Pseudotaxus to Taxus and the inclusion of Amentotaxus, Austrotaxus, Cephalotaxus and Torreya within Taxaceae. Results are consistent with the taxonomic recognition of nine species: T. baccata, T. brevifolia, T. canadensis, T. cuspidata, T. floridana, T. fuana, T. globosa, T. sumatrana and T. wallichiana, but evidence is found for less species distinction and considerable reticulation within the T. baccata, T. canadensis and T. cuspidata group. We compare the results to known taxonomy, biogeography, present new leaf anatomical data and discuss the origins of the hybrids T. ×media and T. ×hunnewelliana.