Analysis of the ploidy levels between different plant species can help the breeder to select strategies for cross-based breeding programs. In this study, various ecotypes of Festuca pratensis, F. arundinacea, F. rubra, Lolium perenne, L. multiflorum, and L. hybridum were cytologically characterized and grouped based on their chromosomal and karyotypic parameters. Moreover, 5S and 45S rDNA loci were determined by fluorescence in situ hybridization (FISH) in four species. Based on the mitotic chromosome complements, all the F. arundinacea and F. rubra ecotypes were hexaploid, L. multiflorum ecotypes were tetraploid and both diploid and tetraploid ecotypes of L. perenne and L. hybridum were identified. All ecotypes were located in the 2A class of Stebbins’s asymmetry, except for Fa2 (F. arundinacea) from Iran, Fr1 (F. rubra) from Finland, and Lp3 (L. perenne) from Switzerland, which was classified as 2B. One or two B chromosomes were observed in several individuals of an Iranian F. pratensis. In a cluster analysis based on karyotype parameters, Lp2 (L. perenne) from Switzerland and Fp2 (F. pratensis) from Sweden showed the minimum distance, suggesting that their crossing might be successful. FISH identified three 5S and two 45S rDNA loci in F. arundinacea, one 5S, and one 45S rDNA loci in F. pratensis, three 5S and two 45S rDNA loci in F. rubra, and one 5S and three 45S rDNA loci in L. hybridum.
