Genomic DNA isolation in cotton is complicated because of the presence of secondary metabolites that are inhibitory to PCR amplification. We report here that radicle tips, but not other parts of cotton seedlings, yield high-quality DNA that is readily amenable for PCR. The radicle-tip-excised seedlings retain viability because of the formation of adventitious roots. We demonstrate the utility of this method in distinguishing homozygotes from heterozygotes in a cotton breeding population and in hybrid seed purity testing.