This document specifies a real-time polymerase chain reaction (real-time PCR) method for the qualitative detection of horse-specific DNA derived from food and feed. It requires the extraction of an adequate amount of PCR amplifiable DNA from the relevant matrix and can be applied to the detection of horse material derived from domestic horse (Equus caballus), mule (Equus caballus ♀ � Equus asinus ♂), hinny (Equus caballus ♂ � Equus asinus ♀) and zebroid (Equus caballus � Equus simplicidens). The assay also detects the species Przewalski's horse (Equus przewalskii) and zebra (Equus burchellii).The target sequence is an Equus caballus isolate Twilight breed thoroughbred chromosome 28, EquCab3.0, whole genome shotgun sequence (i.e. GenBank accession number NC_009171.3), which is present as a single copy per haploid genome. The provided PCR assay for this target has an absolute limit of detection of five copies per reaction, with ≥ 95 % replicability at this concentration (LOD95 %).