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Somaclones, regenerated from Pearl millet cultivar 5141B, were evaluated for various biochemical and molecular changes. Plants (R0) regenerated through tissue culture were evaluated. Biochemical and genetic variation among the somaclones was evaluated by employing DNA- based marker techniques, methylation analysis and RAPD pattern. High frequency of variation in the protein profile was observed. Somaclonal variants analyzed were characterized either by the presence of new polypeptide or by lack of parental type polypeptides. RAPD was carried out with 9 decanucleotide primers. RAPD analysis revealed distinct identity and differentiation for each somaclone and many novel bands were obtained which were not observed in the parental. DNA methylation was evaluated with Sma I, XmaI, Hpa II and Msp I. Variation in the pattern of digestion of the somaclones by these enzymes indicated gross changes in methylation of the bases in the DNA. Also DNA content in individual plant was analyzed. It showed differences in total content indicated existence of ploidy among regenerant. Study consolidates the characterization of clones derived from cell culture. However it needs further characterization to understand changes in somaclonal variants. Key words: Tissue culture, Somaclone, Pearl Millet, Plant regeneration, RAPD, DNA methylation.