C-banded pattern in two accessions of Thinopyrum bessarabicum (Save ex Rayss) A Löve (2n=2x=14, EbEb) and their idiogram was established. C-banding analysis was further used to identify the chromosomes of Tritipyrum amphiploid (2n=6x=42, AABBEbEb) and a BC1F2 genotype from wheat and Tritipyrum. Two 18S-26S rDNA loci were detected on Th. bessarabicum chromosomes by in situ hybridization using an 18S-26S rDNA probe. Eb chromosomes in Tritipyrum generally were identified by their distinctive C-banding patterns which reflected heterochromatin regions. C-banding procedure resulted in sharp and distinct bands in one or both ends of Eb chromosomes without interval bands. Observed C-bands in Eb genome mainly reflected the telomeric and subtelomeric sequences which also showed more strong signals in genomic in situ hybridization. Results showed the importance of the C-banding technique as a screening tool in identification of addition and substitution lines in the progenies of wheat and Tritipyrum crosses during segregating generations