Elucidating patterns of root growth is essential for a better understanding of the functioning of plant-dominated ecosystems. To this end, reliable and inexpensive methods are required to determine species compositions of root samples containing multiple species. Previous studies use a range of PCR-based approaches, but none have examined a species pool greater than 10 or 30 when evaluating mixed and single species samples, respectively. We present a method that evaluates size differences in fluorescently labelled PCR amplicons (fluorescent fragment length polymorphism) of the trnL intron and the trnT-trnL and trnL-trnF intergenic spacers. Amplification success of the trnT-trnL spacer was limited, but variation in the trnL intron and the trnL-trnF spacer was sufficient to distinguish over 80% of the 95 species (97% of the 77 genera) evaluated from a diverse fescue grassland community. Moreover, we identified species known to be present in mixed samples of 4, 8, 12, and 16 species on average 82% of the time. However, this approach is sensitive to detecting species known to be absent (false positives) when using our key of 95 species. Comparing unknowns to a limited species pool ameliorates this problem, comparable to a researcher using prior knowledge of what species could be found in a sample to constrain the identification of species. Comparisons to other methods and future improvements are discussed. This method is efficient, cost- effective and broadly applicable to many ecosystems. © 2010 Blackwell Publishing Ltd.