DNA genotyping is an essential tool used by plant geneticists and plant breeding programs. With the continued cost reduction and increased data output of DNA sequencing technology, it is now feasible to genotype plants by whole genome re-sequencing. This method has been applied successfully to genotype mapping populations in arabidopsis and rice. Although this approach is limited in plants with large unsequenced genomes such as barley, as the sequence coverage obtained per sequencing run is still too low to confidently assess polymorphisms. To address this issue, here we describe the development of a genotyping by targeted genome sequencing approach. The method is based on selectively amplifying genic regions of the genome in a single reaction followed by sequencing and scoring polymorphic bases. The use of DNA barcodes allows the multiplexing of samples in a single sequencing run.