Advances in next-generation technologies have driven the costs of DNA sequencing down to the point that genotyping-by-sequencing (GBS) is now feasible for high diversity, large genome species. We have developed a procedure for constructing GBS libraries based on reducing genome complexity with restriction enzymes (REs). This approach is simple, quick, specific, reproducible, highly scalable and may reach important regions of the genome that are inaccessible to sequence capture approaches. Over the past year, we have applied this technology to a variety of different species including plants (maize, teosinte, sorghum, rice, barley, cassava, cacao, grape, shrub willow) and animals (deer mouse, vole, goose, scrub jay, fox, cow, solitary bee). Data analysis pipelines have also been developed to allow application to species, both with and without sequenced genomes. Future application of GBS to breeding, conservation, and global species and population surveys may allow plant breeders to conduct genomic selection on a novel germplasm or species without first having to develop any prior molecular tools, or conservation biologists to determine population structure without prior knowledge of the genome or diversity in the species.