Although molecular techniques have been used to assess the genetic diversity of some species of Trifolium L. or within the genus generally, there is no record of using these techniques to develop core collections within the genus or evaluate genetic diversity of T. spumosum L. Our study aimed to develop a two step approach for choosing a core collection of T. spumosum as a model approach for other pasture legumes. First, a total of 317 accessions, with near-complete ecogeographical data, were selected from the Australian ex situ collection of T. spumosum. Missing data, including details of geographic ditribution were completed. A preliminary core of about 30% of the collection was, then, selected.

Fluorescent AFLP (fAFLP) was applied for molecular screening of genetic diversity in the collection’s first subset. Four of the most ecogeographically diverse accessions were screened in search of the primers with the highest number of bands and polymorphism. By combining the results of this screening and ecogeographical data we developed the final core collection of 32 accessions, which contains 30% of the first subset.

Our study demonstrated that a combination of AFLP markers and ecogeographical data can be used to develop an effective core collection that maintains most of the genetic diversity of and represents the original collection. Such a core will allow breeders to more effectively select cohorts for field testing and enable gene bank managers to more efficiently conserve germplasm. It will also help identify gaps in genetic diversity, resulting in a more effectively targeted collection in future. The quality of ecogeographical data of collection sites is critical to the success of this approach to developing a core collection. This model can be expanded to sampling accessions for phylogenetic studies of other crops and native plants with large collections.