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Ispaghula (Plantago ovata) is used to reduce gastrointestinal and urinary tract infections, as well as control blood glucose and cholesterol levels in the human body. The phylogeny and genetic diversity of 22 different ecotypes of Ispaghula were evaluated using 12 ISSR markers and nine morphological and phenological traits. Analysis of variance showed that there were significant differences among cultivars for all traits. Cluster analysis grouped 22 different ecotypes of Ispaghula in two groups using UPGMA method based on field data. The assessment of genetic diversity among ecotypes based molecular markers showed that the 12 primers amplified 91 polymorphic bands. The maximum number of bands (11) was produced by UBC813 and primers UBC811 with 10 bands were in the next steps, respectively. The minimum band number (4) was produced by UBC824. Polymorphism information content (PIC) value was varied from 0.26 to 0.45 and Marker index (MI) was varied from 0.90 to 4.13. Cluster analysis using UPGMA based on molecular markers, placed 22 ecotypes in the study in five groups, include 1, 1, 2, 3 and 15 ecotypes, respectively. Grouping of ecotypes with molecular markers was different with classification of the ecotypes based morphological traits. According to the results, ecotypes that are far apart can be used in the breeding program of Ispaghula.