Planago ovata L. is an economically important species in the monotypic genus Plantago. It is a short-stemmed annual herb. The seed husk of this plant is commonly called psyllium or isabgol which is important in pharmaceutical formulation and food industry. In this study, callus induction was optimized using different explants of Plantago ovata. Callus DNA was utilized to access the somaclonal variations using the Random Amplification of Polymorphic DNA (RAPD) markers. The maximum callus growth was observed in Murashige and Skoog (MS) medium containing 4 mg $L^{-1}$ 2,4-D concentration for shoots, 0.5 mg $L^{-1}$ for seeds and 2 mg $L^{-1}$ for roots. Moreover, the effect of culture age was considered in assessing genetic variability. Maximum genetic variability was observed in the DNA samples of callus at the concentration of 2 mg $L^{-1}$ 2,4-D for all explants (roots, shoots, and seeds). Cluster analysis was performed based on 1) similarity coefficient between samples and 2) molecular data using the Numerical Taxonomy and Multivariate Analysis System (NTSYS) PC version 2.01; similarity index was generated by similarity for Quantitative Data (SIMQUAL). Our study indicated that Random Amplified Polymorphic DNAs can successfully be used to explore polymorphism among callus samples at different hormonal concentrations. This study can be useful for the production of callus from Plantago ovata and estimation of genetic variations due to tissue culture conditions. Evaluation of genetic variations can display novel features and manipulate genetic bottlenecks in Plantago ovata. New genetic variations in somaclones can bring vital insight for plant improvement.