Sainfoin (Onobrychis viciifolia Scop.) is a perennial forage legume widely distributed in the northern temperate regions of the world. Its genetic improvement has been relatively slow due, in part, to the lack of genetic information including molecular characterization of sainfoin germplasm. An attempt was made to evaluate genetic diversity and relationships among 38 sainfoin accessions collected from different regions of the world using amplified fragment length polymorphism (AFLP) markers. Five AFLP primer pairs were used to assess 367 individual plants, which produced 1042 polymorphic AFLP bands. The frequencies of the scored bands in all assayed individuals ranged from 0.003 to 0.973, with a mean value of 0.165. The analysis of molecular variance revealed higher within-accession (84.3%) genetic variation than among accessions (15.7%). The genetic distance based on inter-accession distance matrices was significant for most accessions but was not significant for accessions sharing similar parents. A dendrogram of the collected accessions showed two clusters at an inter-accession genetic distance coefficient of 0.36. The revealed information on genetic distance and genetic diversity of the sainfoin accessions is useful for selecting genetically diverse germplasms for sainfoin genetic improvement efforts.