Key message We provide evidence that nucleotide sequence and methylation status changes occur in the Arabidopsis genome during in vitro tissue culture at a frequency high enough to represent an important source of variation. Somaclonal variation is a general consequence of the tissue culture process that has to be analyzed specifically when regenerated plants are obtained in any plant species. Currently, there are few studies about the variability comprising sequence changes and methylation status at the DNA level, generated by the culture of A. thaliana cells and tissues. In this work, two types of highly reproducible molecular markers, modified methylation sensitive AFLP (metAFLP) and transposon methylation display (TMD) have been used for the first time in this species to analyze the nucleotide and cytosine methylation changes induced by transformation and tissue culture protocols. We found significantly higher average methylation values (7.5%) in regenerated and transgenic plants when compared to values obtained from seed derived plants (3.2%) and that the main component of the somaclonal variation present in Arabidopsis clonal plants is genetic rather than epigenetic. However, we have found that the Arabidopsis regenerated and transgenic plants had a higher number of non-fully methylated sites flanking transposable elements than the control plants, and therefore, their mobilization can be facilitated. These data provide further evidence that changes in nucleotide sequence and methylation status occur in the Arabidopsis genome during in vitro tissue culture frequently enough to be an important source of variation in this species.