Next-generation sequencing technologies have a massive throughput, which dramatically reduces the cost of sequencing per gigabase, compared to standard Sanger sequencing. To make the most efficient use of this throughput when sequencing small regions or genomes, we developed a barcoding method, which allows multiplexing of 96 or more samples per lane. The method employs 8 bp tags, incorporated into each sequencing library during the library preparation enrichment polymerase chain reaction (PCR), pooling bar-coded libraries in equimolar ratios based on quantitative PCR, and sequencing using the three-read Illumina method.