Diversity Arrays Technology (DArT™) was evaluated as a tool for determining molecular diversity of wheat landraces held within the Australian Winter Cereals Collection (AWCC). Initially, a set of 44 wheat landraces was evaluated with 256 DArT markers. The dataset was compared with the results obtained using 16 amplified fragment length polymorphism (AFLP) primer combinations and 63 simple sequence repeat (SSR) markers, mapped on the 21 chromosomes, from a previous study. The DArT markers exhibited a strong positive correlation with AFLP and SSR, with each marker type distinguishing similar relationships among the 44 landrace accessions. The DArT markers exhibited a higher polymorphic information content than AFLP, and were comparable with that obtained with SSR. Three hundred and fifty-five DArT markers were then used to evaluate genetic diversity among 705 wheat landrace accessions from within the AWCC, chosen to represent 5 world regions. DArT analysis was capable of distinguishing accessions from different geographic regions, and suggested that accessions originating from Nepal represent a unique gene pool within the collection. A statistical resampling of DArT loci indicated that 10–20 loci were enough to distinguish the maximum molecular diversity present within the collection. This research demonstrates the efficacy of the DArT platform as a tool for efficient examination of wheat diversity. As an ex situ germplasm repository, the AWCC contains wheat accessions of high genetic diversity, from genetically differentiated collection sites, even though diversity was under-represented in some countries represented in the repository.