Seven wheat-Thinopyrum amphiploids, AT 3425, AgCs, PI 550710, PI 550711, PI 550712, PI 550713 and PI 550714, were evaluated for perennial growth habit in the field. Three of them, AgCs, AT 3425, and PI 550713, were identified as perennials. Fluorescent genomic in situ hybridization (FGISH) patterns of mitotic chromosomes indicated that AgCs had seven pairs of Thinopyrum chromosomes and 21 pairs of wheat chromosomes. PI 550713 and AT 3425 showed similar FGISH patterns of mitotic chromosomes with three pairs of wheat-Thinopyrum translocated chromosomes, seven pairs of Thinopyrum chromosomes, and 18 pairs of wheat chromosomes. Thinopyrum chromosome pairing in the Fi hybrid of AT 3425 with AgCs demonstrated differences between Thinopyrum genomes in these two amphiploids. Based on chromosome constitutions, pairing and reported pedigrees, AgCs and AT 3425 were identified as a wheat-Thinopyrum elongatum amphiploid and partial wheat-Thinopyrum ponticum amphiploid, respectively. Chromosome pairing in the F1 hybrid between AT 3425 and PI 550713 revealed that these two amphiploids contained the same Thinopyrum genome. Two different Thinopyrum genomes conferring perennial growth habit were identified from the perennial amphiploids and characterized cytogenetically.