We evaluated the genome structure and salt stress response of BC1F6 lines derived from a cross between an Iranian slat tolerant bread wheat cultivar ʻRoushanʼ (2n = 6x = 42, AABBDD) and a synthetic allohexaploid Tritipyrum ʻKabCrbʼ (2n = 6x = 42, AABBEbEb). Sixteen lines (L1 to L16) and their parents were studied for their chromosome composition, K+:Na+ ratio and relative dry mass (RDM) by growing them in nutrient solution containing 0 (control) or 100 mM NaCl (treatment). 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 L3, L4, L7, L8, L14, L15 and L16 were DS6Eb(6D). There were high differences in morphological traits and salinity tolerance even among DS6Eb(6D) substitutions or untranslocated lines maybe due to unidentified rearrangement or small introgressions.