Hybrids between hexaploid wheat (Triticum aestivum) and the tall wheatgrass Thinopyrum ponticum are valuable for the transfer of traits such as disease resistance and for perennial wheat development. Through crosses between wheat and Th. ponticum, we have created several hybrid lines of unknown chromosome constitution. This study aims to characterize their total chromosome number and origin. Six advanced hybrid lines were chosen for chromosome characterization based on their morphological diversity. The diploid chromosome number of each plant was determined by counting chromosomes in young root tips. The genome composition was determined using genomic in situ hybridization (GISH) and C-banding to distinguish between the wheat and wheatgrass genomes and identify specific chromosomes. Cross compatibility studies between the hybrid lines were also undertaken to determine fertility of their F1 progeny and to confirm differences in their chromosome constitution as indicated by chromosome pairing during meiosis. Some lines vary with regard to chromosome number and composition. There some compatibility between the lines as indicated by seed set, fertility of the F1 offspring and pairing of the parental chromosomes during meiosis of the F1 offspring. Identification of common chromosomes involved in a perennial habit for wheat will indicate their necessity in plant functioning and maintaining a polycarpic lifecycle.