Tissue culture is used as a tool to propagate seedlings massively in industry. However, unfavorable genomic alteration that affects phenotypic characters occurs in the procedure. A technique which scans these genetic changes genome-wide can enables us to discriminate the clone that has genomic alteration at early stages. In this study, we used restriction landmark genome scanning (RLGS) to detect alterations among ramets of rice, which redifferentiated from a callus that had been cultured for 4 weeks. As a result, 6% (10 spots) of the total (161 spots examined) were different between two ramets. One spot out of ten was cloned and was located on a predicted gene. Application of a PCR-based method to the RLGS spot region detected one methylation alteration among the ramets. Accordingly, RLGS was successful in surveying methylation alteration events during tissue culture in plants and it should be possible to use this method for monitoring of alteration levels.