The last decade has seen a number of studies reporting the extraction of DNA from ancient sources, such as fossil bones. Owl pellets, which contain an excellent skeletal record of small mammals consumed, can be used in a non-invasive sampling method for genetic studies of free-ranging animals without the need for direct capture or even observation. Such a non-invasive sampling method will allow us to address questions that cannot be answered using conventional methods and will lead to a more integrated study of micromammals. In the present study, various protocols used for ancient DNA extraction were investigated, in order to determine the applicability of owl pellets as a source of DNA for phylogenetic and phylogeographical studies of micromammals. Of the 12 bone samples used in this study, 11 gave sequences of expected species (Crocidura, Rattus and Mus) and size (around 300 bp), using the pair of primers L14841/H15149, which target a highly conserved region of the cytochrome b gene. The results obtained demonstrate that mitochondrial DNA can be isolated and amplified using bones of micromammals found in barn owl (Tyto alba) pellets. The recovery of genetic data from owl pellets will enable the identification of prey species for several phylogenetic and phylogeographical studies of small mammals (Muridae and Insectivora) appearing in the owl's diet. The main advantages of this new approach are that (a) the pellets are very easy to find and collect, (b) the pellets can potentially provide a large number of individuals of small mammals, and (c) the method can cover a wide geographical area complementary to the range of owls of this type. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 85, 331–340.