Abstract"Replanting disease" is a serious constraint to root growth in the medicinal species Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. and C.A. Mey. The syndrome involves an array of morphological, physiological and biochemical changes to the plant, which culminates in a major loss in tuberous root growth. Here, the tendency of replanting disease to induce differential cytosine methylation in the root DNA was explored via the methylation-sensitive amplified polymorphism (MSAP) method. Exposure to the disease measurably altered the global methylation level. Of the 231 differentially methylated MSAP fragments identified, 136 involved replanting disease-induced methylation and 95 demethylation. A set of 31 differentially methylated fragments was isolated and sequenced. The sequences were used to analyze the function of the genes involved and to investigate whether any were differentially transcribed as a result of exposure to replanting disease. Of the eight genes subjected to transcription profiling, the three which were demethylated in the diseased roots were transcribed more abundantly in these roots, and the five which were methylated were down-regulated by a real-time quantitative PCR (qPCR) method. Our study gives an insight into the DNA methylation of R. glutinosa subjected to replanting disease and provides valuable information for further exploring epigenetic regulation of responses to the disease in the species and other plants.