We developed a multiplex PCR DNA marker for quick and easy identification of the AAGG-genome timopheevii lineage, including Triticum timopheevii, Triticum araraticum and hexaploid Triticum zhukovskyi (AAAmAmGG), and the AABB-genome emmer wheat lineage, including Triticum durum, Triticum dicoccum and Triticum dicoccoides. Distinguishing between tetraploid AAGG- and AABB-genome wheat species based on morphology is known to be difficult. This multiplex PCR system is based on the simultaneous PCR amplification of two chloroplast regions, matK and rbcL. The matK region molecularly distinguishes the two lineages, whereas the rbcL region is a positive control amplicon. We also examined whether the simple sequence repeat is a fixed mutation within species, using genetic resources in the collection of KOMUGI, Kyoto University, which comprises accessioned species collected across diverse geographical areas. The multiplex PCR marker distinguished AAGG from AABB species with complete accuracy.