Horses, asses and zebras, can produce first-generation Fl-hybrids, despite their striking karyotypic and phenotypic differences. Such Fl-hybrids are mostly infertile, but often present characters of considerable interest to breeders. They were extremely valued in antiquity, and commonly represented in art and on coinage. However, hybrids appear relatively rarely in archaeological faunal assemblages, mostly because identification based on morphometric data alone is extremely difficult. Here, we developed a methodological framework that exploits high-throughput sequencing data retrieved from archaeological material to identify Fl-equine hybrids. Our computational methodology is distributed in the open-source Zonkey pipeline, now part of PALEOMIX (https://github.com/MikkelSchubert/paleomix), together with full documentation and examples. Using both synthetic and real sequence datasets, from living and ancient Fl-hybrids, we find that Zonkey shows high sensitivity and specificity, even with limited sequencing efforts. Zonkey is thus well suited to the identification of equine Fl-hybrids in the archaeological record, even in cases where DNA preservation is limited. Zonlcey can also help determine the sex of ancient animals, and allows species identification, which advantageously complements morphological data in cases where material is fragmentary and/or multiple candidate equine species coexisted in sympatry. (C) 2016 Elsevier Ltd. All rights reserved.