The emergence of new sequencing technologies has provided fast and cost-efficient strategies for high-resolution mapping of complex genomes. Although these approaches hold great promise to accelerate genome analysis, their application in studying genetic variation in wheat has been hindered by the complexity of its polyploid genome. Here, we applied the next-generation sequencing of a wheat doubled-haploid mapping population for high-resolution gene mapping and tested its utility for ordering shotgun sequence contigs of a flow-sorted wheat chromosome. A bioinformatical pipeline was developed for reliable variant analysis of sequence data generated for polyploid wheat mapping populations. The results of variant mapping were consistent with the results obtained using the wheat 9000 SNP iSelect assay. A reference map of the wheat genome integrating 2740 gene-associated single-nucleotide polymorphisms from the wheat iSelect assay, 1351 diversity array technology, 118 simple sequence repeat/sequence-tagged sites, and 416,856 genotyping-by-sequencing markers was developed. By analyzing the sequenced megabase-size regions of the wheat genome we showed that mapped markers are located within 40-100 kb from genes providing a possibility for high-resolution mapping at the level of a single gene. In our population, gene loci controlling a seed color phenotype cosegregated with 2459 markers including one that was located within the red seed color gene. We demonstrate that the high-density reference map presented here is a useful resource for gene mapping and linking physical and genetic maps of the wheat genome.