Sainfoin (Onobrychis viciifolia Scop.) is a perennial forage legume that possesses beneficial properties in the context of sustainable agriculture. In order to initiate a pre-breeding programme, we have assembled a germplasm collection of O. viciifolia from many geographic regions, as well as a collection of Onobrychis species that might be crossed with O. viciifolia to improve its biological and agronomic properties. The objective of this study was to obtain DNA sequences from a representative sample of these accessions in order to characterise their genetic diversity and to assess the robustness of the Onobrychis taxonomy. Because of potential problems with copurification of tannins and polyphenols from Onobrychis leaf tissue, three methods for DNA extraction were assessed and the most appropriate one identified. DNA sequences were obtained for the nuclear internal transcribed spacer region and the trnH-psbA and trnT-trnL intergenic spacers of the chloroplast genome. Neighbour joining trees were constructed and accessions were assigned to operational taxonomic units. The results indicated that there is substantial genetic diversity among Onobrychis species. Redundancies in species nomenclature were identified, as well as possible overlap between some of the sections into which the Onobrychis genus has been divided. A genetic distinction was apparent between O. viciifolia accessions from western Europe and those from eastern Europe and Asia, reflecting a similar division based on agronomic properties. Recognition of these different though related germplasms will be valuable in the design of breeding programmes for the rational improvement of sainfoin as a forage crop for sustainable agriculture.