Fine roots of trees are intensively used as indicators to assess soil alterations, e.g. those owing to atmospheric inputs of acidifying substances, but their identification to species with morphological criteria is difficult. In this study, we established molecular techniques in order to identify fine roots of the 30 most common tree species of the Alps. We developed a protocol for efficient isolation of DNA from fine roots with extraction of DNA in the presence of polyvinylpyrrolidone (PVP) and polyvinylpolypyrrolidone (PVPP). The trnL (UAA) intron of plastid DNA was used as a marker for fine root identification. We amplified and sequenced this intron with plant universal primers. The size of the sequences ranged from 444 to 672 bp. A synoptic key for species identification was designed on the basis of restriction fragment patterns predicted from sequence data. Using the restriction enzyme TaqI as key enzyme, and where necessary HinfI, RsaI and CfoI, 16 taxa, including Picea abies, Larix decidua, Abies alba, and Fagus sylvatica, the dominant tree species of the Alpine region could be identified by agarose gel electrophoresis of restriction fragments. Fourteen taxa could be identified to the genus level, among them Quercus, Salix and Populus species. In a field study, conducted in a 20 × 30 m plot of a mixed forest with five tree species, fine roots of 43 out of 46 samples were identified and their distributions were mapped. These results demonstrate the utility of our DNA extraction method and of the trnL intron for the identification of fine tree roots.