The aim of this study was to make morphological and molecular characterization of tall wheatgrass (Thinopyrum ponticum) accessions naturally found in Canakkale flora, Turkey. The seeds collected from 24 different locations in Canakkale vicinity were planted in the nursery field to determine thirteen morphological parameters. Twelve universal rice primers (URPs) were used to reveal genetic relationship among the accessions while ploidy analysis was done based on the nuclear DNA content of plants determined by Flow cytometer. Fluorescence in situ hybridization (FISH) method was used to determine 5S and 45S rDNA distributions on mitotic chromosomes. The agro-morphological data showed significant variation among the accessions for all parameters measured, except the number of nodes per plant. Twelve URP primers produced 73 alleles in total and 63 of those were polymorphic. The highest polymorphism information content value was obtained from URP 17R with 0.82. The first three components of Eigen values in PCA analysis explained 41.1% of total variation. The 2C nuclear DNA contents of the accessions ranged from 41.17 to 45.49 pg. All the accessions were determined as decaploid with 2n = 70 chromosomes. FISH analysis provided 18-20 interstial 5S rDNA and 12-14 terminal 45S rDNA loci. The results concluded that tall wheatgrass accessions used in this study contain a significant variation in morphological traits and PCR-based DNA polymorphism which could be used as a new genetic resource in breeding programs of tall wheatgrass and wheat improvement for intra- and intergeneric crosses.