We describe 2b-RAD, a streamlined restriction site-associated DNA (RAD) genotyping method based on sequencing the uniform fragments produced by type IIB restriction endonucleases. Well-studied accessions of Arabidopsis thaliana were genotyped to validate the method's accuracy and to demonstrate fine-tuning of marker density as needed. The simplicity of the 2b-RAD protocol makes it particularly suitable for high-throughput genotyping as required for linkage mapping and profiling genetic variation in natural populations.