Genetic relationships among 154 genotypes, including 50 species, held within the UK National Willow Collection were analysed using nine primer combinations in an optimised fluorescent amplified fragment length polymorphism (AFLP®) protocol. The AFLP® data resolved relationships at all levels, from discriminating between closely related accessions to differentiating among majority of species, sections and subgenera. The neighbourjoining dendrogram split accessions into three major wellsupported clusters, two of which comprised species of the subgenera Vetrix and Salix. Surprisingly, the third (98% bootstrap support) comprised only Salix triandra accessions. The genetic similarity (GS) between S. triandra and Salix or Vetrix was similar (0.39 and 0.40, respectively) and greater than the genetic similarity between Salix and Vetrix (GS=0.57). Separate clustering of S. triandra is also supported by hierarchical analysis of molecular variance (AMOVA), that partitioned 31.4% of the total variance between these three groups, whereas only 16.3% was partitioned between the two subgenera. These results challenge all current classifications which assign S. triandra to subgenus Salix. Principal coordinate analysis gave corresponding results and facilitated interpretation of relationships among species within sections of the two subgenera, which are discussed. The study included 40 species which have been used in breeding, and the findings will facilitate the choice of parents and interpretation of the results of different crosses, on the basis of more accurate knowledge of genetic relationships. AFLPs® also detected identical genotypes (within the limits of AFLP® error) which should not be used as distinct parents in breeding programmes.