Question: Is it possible to determine the species composition of root samples containing multiple species, without first disentangling individual roots? Methods: The internal transcribed spacer (ITS) region of nuclear ribosomal DNA was amplified and sequenced from four California annual grassland species (two Poaceae and two Asteraceae). Restriction enzymes that cut the ITS region of each species into uniquely sized fragments were identified based on DNA sequence variation of the ITS regions. Mixed root samples were analysed to test the ability of the method to identify the presence or absence of each species in multispecies samples.Results: The technique successfully identified species present in multi-species samples. ITS regions were shorter in Poaceae than in Asteraceae, so size differences alone were sufficient to distinguish these taxonomic groups. At the species level, digestion of ITS regions with the appropriate restriction enzymes yielded at least one uniquely sized fragment for each species. Conclusions: This method is the first to identify the species composition of mixed root samples. It should be applicable to most plant species because the ITS region is flanked by universal primers and most species have unique ITS sequences. The ability to determine species-specific rooting distributions has broad applications in vegetation science.