Helianthus tuberosus L. is regarded as one of the most important bioenergy crops because of its tolerance to arid conditions and its high biomass production. Future breeding programmes will rely on the available germplasm, thus necessitating a critical assessment of genetic diversity and differentiation in the species. The germplasm diversity and regional differentiation of H. tuberosus L. was assessed for 60 accessions collected from East Asia and Europe by means of amplified fragment length polymorphisms (AFLPs), phenotypic traits and chemical analysis. The analysis did not reveal separate clusters for accessions from East Asia and Europe, with 5% for genetic and 0·27% for phenotypic variability, although some regional accessions were closely related to each other with respect to morphological, chemical and genetic variation. Both phenotypic and genetic relationships showed a moderate correlation with colour of tubers, which can be used as an important criterion for germplasm management of the crop. Three major genetic groups were identified from the accessions. Within the groups derived from the genetic data, both morphological and chemical traits were very variable. The most important features of Groups I, II and III were: Group I, strong sexual reproduction, higher above-ground biomass and nutrient content; Group II, longer vegetative growth; Group III, higher tuber yield and total sugar content. High diversity was found in both European and East Asian accessions as well as within each genetic group, suggesting that there is a strong base for future breeding from these plants.