Belowground vertical community composition and maximum rooting depth of the Edwards Plateau of central Texas were determined using DNA sequence variation to identify roots from caves 5 to 65 m deep. Roots from caves were identified by comparing their DNA sequences for the internal transcribed spacer (ITS) region of the 18S-26S ribosomal DNA repeat against a reference ITS database developed for woody plants of the region. Sequencing the ITS provides, to our knowledge, the first universal method for identifying plant roots. At least six tree species in the system grew roots deeper than 5 m, but only the evergreen oak, Quercus fusiformis, was found below 10 m. The maximum rooting depth for the ecosystem was approximately 25 m. 18O isotopic signatures for stemwater of Q. fusiformis confirmed water uptake from 18 m underground. The availability of resources at depth, coupled with small surface pools of water and nutrients, may explain the occurrence of deep roots in this and other systems.