PCR has become one of the most popular techniques in functional genomics. Projects in both forward and reverse genetics routinely require PCR amplification of thousands of samples. Processing samples to extract DNA of sufficient purity for PCR is often a limiting step. We have developed a simple 96-well plate-based high-throughput DNA extraction method that is applicable to many plant species. The method involves a simple incubation of plant tissue samples in a DNA extraction buffer followed by a neutralization step. With the addition of a modified PCR buffer, the extracted DNA enabled the robust amplification of genomic fragments from samples of Arabidopsis, tobacco, sorghum, cotton, moss, and even pine needles. Several thousand DNA samples can be economically processed in a single day by one person without the use of robotics. This procedure will facilitate many technologies including high-throughput genotyping, map-based cloning, and identification of T-DNA or transposon-tagged mutants for known gene sequences.