The use of transgenic crops is steadily increasing around the world, led by soybean (based on total area) and maize (in terms of total number of countries). Transgenic maize is grown in at least 17 countries across four continents: Africa, America, Asia and Europe. The comprehensive global spread of transgenic maize has significant implications for organizations involved in germplasm conservation and genetic enhancement; particularly as some countries require a GMO-free declaration when receiving shipments of maize germplasm. This article describes the protocol used by the International Maize and Wheat Improvement Center (CIMMYT) for monitoring unintentional transgene flow in maize genebank and breeding plots. The protocol is based on polymerase chain reaction (PCR) markers for detecting specific recombinant DNA sequences in bulked samples collected from sentinel plots. To date, no unintentional transgene flow has been detected in CIMMYT fields of maize genebank accessions or breeding materials.