Previous tribal classifications of Rhamnaceae have been based on fruit characters, resulting in the delimitation of large and otherwise heterogeneous groups. We evaluated the most recent classification with DNA sequences of two regions of the plastid genome, rbcL and trnL-F, from 42 genera of Rhamnaceae and representatives of the related families Elaeagnaceae, Barbeyaceae, Dirachmaceae, Urticaceae, Ulmaceae, Moraceae, and Rosaceae. The trnL-F trees have higher consistency and retention indices than the rbcL trees, and patterns of change in rbcL and trnL-F are compared. The closest relatives of Rhamnaceae are Dirachmaceae and Barbeyaceae, followed by the urticalean families. The plastid trees support the monophyly of the family and provide the basis for a new tribal classification. Three strongly supported clades are identified, but morphological characters could not be found to underpin a formal taxonomic description of these three clades as subfamilies. We therefore only recognize groups that are also defined by morphological characters. The biogeography of Rhamnaceae is discussed with reference to the molecular trees.