Using next-generation sequencing technologies it is possible to resequence entire plant genomes or sample entire transcriptomes more efficiently and economically and in greater depth than ever before. Rather than sequencing individual genomes, we envision the sequencing of hundreds or even thousands of related genomes to sample genetic diversity within and between germplasm pools. Identification and tracking of genetic variation are now so efficient and precise that thousands of variants can be tracked within large populations. In this review, we outline some important areas such as the large-scale development of molecular markers for linkage mapping, association mapping, wide crosses and alien introgression, epigenetic modifications, transcript profiling, population genetics and de novo genome/organellar genome assembly for which these technologies are expected to advance crop genetics and breeding, leading to crop improvement.