DNA sequencing technology is advancing at an astounding rate, with rapid increases in data volumes and quality combined with reducing costs. The availability of this technology opens novel avenues for the analysis of plant germplasm resources. Where previous studies analysed a limited number of phenotypic or molecular genetic markers, it is now possible to re-sequence whole genomes to characterise diversity at a resolution of each nucleotide. Current approaches combine high resolution genetic markers with genome sequencing both for reference assembly and genotyping by sequencing. As next generation sequencing technologies continue to advance, we approach the potential to catalogue and characterise all genome variations across diverse germplasm to gain a greater understanding of how the genome contributes to the diversity seen in today’s plants.