A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species is described. This method uses restriction enzymes (REs) to reduce genome complexity and generate sequence tags for genotyping. The approach is simple, quick, highly specific, and reproducible, allowing access to regions of the genome that are difficult to reach with other methods. Methylation-sensitive REs help avoid repetitive regions and target lower copy regions more efficiently. This simplifies computational alignment challenges in species with high genetic diversity. The method was tested on maize and barley populations, generating tens of thousands of sequence tags. For species without a complete genome sequence, a reference map can be developed around restriction sites, or sequence tags can be treated as dominant markers. The GBS approach is cost-effective and suitable for population studies, breeding, and conservation. It allows genomic selection without prior molecular tools and population structure analysis without prior genome knowledge. The method uses next-generation sequencing with barcoded adapters to enable multiplex sequencing, reducing costs and improving efficiency. The approach is adaptable to various species and has potential applications in breeding, conservation, and global species surveys. The method involves DNA extraction, restriction enzyme digestion, adapter ligation, and sequencing. Data filtering and mapping were performed to identify SNPs and genetic markers. The results show high-quality sequence tags with good mapping accuracy. The GBS approach is efficient, cost-effective, and suitable for high diversity species, offering a valuable tool for genetic studies.A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species is described. This method uses restriction enzymes (REs) to reduce genome complexity and generate sequence tags for genotyping. The approach is simple, quick, highly specific, and reproducible, allowing access to regions of the genome that are difficult to reach with other methods. Methylation-sensitive REs help avoid repetitive regions and target lower copy regions more efficiently. This simplifies computational alignment challenges in species with high genetic diversity. The method was tested on maize and barley populations, generating tens of thousands of sequence tags. For species without a complete genome sequence, a reference map can be developed around restriction sites, or sequence tags can be treated as dominant markers. The GBS approach is cost-effective and suitable for population studies, breeding, and conservation. It allows genomic selection without prior molecular tools and population structure analysis without prior genome knowledge. The method uses next-generation sequencing with barcoded adapters to enable multiplex sequencing, reducing costs and improving efficiency. The approach is adaptable to various species and has potential applications in breeding, conservation, and global species surveys. The method involves DNA extraction, restriction enzyme digestion, adapter ligation, and sequencing. Data filtering and mapping were performed to identify SNPs and genetic markers. The results show high-quality sequence tags with good mapping accuracy. The GBS approach is efficient, cost-effective, and suitable for high diversity species, offering a valuable tool for genetic studies.