The best known and most thoroughly studied epigenetic phenomenon is DNA methylation, which plays an important role in regulating gene expression during plant regeneration and development. In this study, the methylation-sensitive amplified polymorphism (MSAP) technique was carried out to determine differences in methylation profiles between two forms of protocorm-like bodies (PLBs), continuously proliferating PLBs (cPLBs) and spontaneously-differenting PLBs (sdPLBs), derived from cultures of Cymbidium hybridium. A total of 72 selective primer combinations were used to assess the status of cytosine methylation of DNA in these tissues. Of 4,440 fragments obtained 911 fragments, each representing a recognition site cleaved by one or both of the isoschizomers (Hpa II and Msp I), were amplified and were significantly different between the two forms of PLBs. Frequency of total and full-methylation of cPLBs and sdPLBs were 26.7/12.2%, 24.1/11.1%, respectively. In addition, 14 types of MSAP patterns detected in the two forms of PLBs belonged to two classes, type I and II. Sequencing of 14 differentially methylated fragments and their subsequent blast search revealed that cytosine methylated 5'-CCGG-3' sequences were equally distributed in the coding and non-coding regions. Southern blotting was conducted to verify the methylation polymorphism.