Genome structure of BC1F6 lines derived from an Iranian bread wheat cultivar Roushan (2n = 6x = 42, AABBDD) and a synthetic allohexaploid Tritipyrum (2n = 6x = 42, AABBEbEb) were analysed. Sixteen lines (L1 to L16) were studied for their chromosome composition. Among the studied lines, eight wheat-Thinopyrum bessarabicum (Eb genome) disomic substitution lines were identified using PCR-based Landmark Unique Gene (PLUG) markers and cytogenetic analysis. Ten (37%) of 27 PLUG markers used, were informative for Eb chromosomes 1, 2, 3, 4 and 6 in wheat background. PLUG markers also revealed that one line L9 was a disomic substitution line DS2Eb (2B) and seven lines were DS6Eb (6D).