A species-specific PCR assay was developed for the detection of low levels of pork, horse and donkey meat in cooked sausages. Oligonucleotid primers were designed for amplification of species-specific mitochondrial DNA sequences of each species and detected the presence of 0.01 ng of template DNA in water. When applying the assay to DNA extracts from sausages samples that were prepared from binary meat mixtures, it was possible to detect each species when spiked in any other species at the 0.1% level. In conclusion, it can be suggested that this assay can be used to determine mislabelled and/or fraudulent species substitution in comminuted meat products. (c) 2007 Elsevier Ltd. All rights reserved.