Karyotypic study of nine populations belonging to five Festuca species showed chromosome numbers of 2n = 2x = 14, 2n = 4x = 28 and 2n = 6x = 42. The chromosome number of F. elwendiana is reported for the first time. The chromosomes were mostly metacentric and sub-metacentric. Significant differences in the size of chromosomes and their arm ratios indicated the occurrence of quantitative changes in the chromatin (DNA) material during species diversification. The species also differed in their karyotypic formulae possibly due to the occurrence of chromosome structural changes. The karyotypic data and meiotic characteristics of the species were used for multivariate analysis, which partly supports taxonomic treatment of the genus Festuca in the flora of Iran.