© 2018 Background: Epigenetic modifications are key factors modulating the expression of genes involved in the synthesis of phytochemicals. The knowledge of plant epigenetic and genetic variations can contribute to enhance the production of bioactive compounds. These issues have been little explored thus far in Rorippa nasturtium var. aquaticum L. (watercress), an edible and medicinal plant. The aim of the current study was to determine and compare the phenolic composition and epigenetic and genetic variations between wild and cultivated watercress. Results: Significant differences were found in the quantitative phenolic composition between wild and cultivated watercress. The eight primer combinations used in the methylation-sensitive amplification polymorphism (MSAP) method revealed different epigenetic status for each watercress type, the cultivated one being the most epigenetically variable. The genetic variability revealed by the EcoRI/MspI amplification profile and also by eight inter-simple sequence repeat (ISSR) primers was different between the two types of watercress. The results of the Mantel test showed that the correlation between genetic and epigenetic variations has diminished in the cultivated type. Cluster analyses showed that the epigenetic and genetic characterizations clearly discriminated between wild and cultivated watercress. Conclusions: Relevant chemical, epigenetic, and genetic differences have emerged between wild and cultivated watercress. These differences can contribute to fingerprint and develop quality control tools for the integral and safety use and the commercialization of watercress. The richness of epialleles could support the development of tools to manipulate the watercress epigenome to develop high bioproduct–producing cultivars. How to cite: Gutiérrez-Velázquez MV, Almaraz-Abarca N, Herrera-Arrieta Y, et al. Comparison of the phenolic contents and the epigenetic and genetic variability of wild and cultivated watercress (Rorippa nasturtium var. aquaticum L.). Electron J Biotechnol 2018;34. https://doi.org/10.1016/j.ejbt.2018.04.005.