Isabgol is an annual herb cultivated as a medicinal plant. In the present study, karyotypic analysis of 12 Iranian endemic Isabgul (Plantago ovata Forsk.) ecotypes carried out. The chromosome number was 2n=2x=8 with 4m+4st karyotype formula. The mean value of total chromosome length (TL) was 2.86 μm, ranging from 2.44 μm (E4) to 3.26 μm (E12). All ecotypes had two pairs of satellites (3S, 4S), varying in size from 0.34 to 0.36 μm. Most of the chromosomal parameters were significantly different among ecotypes. In general, karyotypes were symmetrical and fell in the Stebbins 2A category. The scatter diagram of A1 and A2 asymmetry indicated 4 groups of ecotypes while PCA analysis together with cluster analysis and scatter diagram of TL and CI, showed 3 groups, containing 9, 2 or 1 ecotypes. C-banding pattern and fluorescence in situ hybridization (FISH) with 45S rDNA probe provided landmarks for chromosome identification in somatic preparations