In the present study,DNA methylation specific PCR primers were designed according to the sequences of CpG island region throughout 12 chromosomes and the specific genomic position.In order to investigate the changes of DNA methylation level and pattern in the cold tolerance introgression lines (ILs,IL5335 and IL5423) and their parents,we used these special primers to amplify the previously digested genomic DNA by McrBC enzyme.(1)Analysis of 83 pairs of primers distributed across the 12 chromosomes of rice showed that the frequency of DNA methylation in IL5335 and IL5423 was to 46.6% and 53.8%,respectively,which was lower than that of recurrent parent (Oryza sativa L.cv.Xieqingzao B).Most of DNA methylation patterns of recurrent parent could be inherited in the ILs.At the same time,some of methylation patterns have changed including demethylation (13.3-18.1%) and hypermethylation (4.4%-6.0%).(2)In the QTLs region related to cold tolerance,the frequency of DNA methylation in IL5335 and IL5423 was 13.3%-26.7%,which was much lower than that of recurrent parent.The frequency of DNA methylation patterns changes of the two ILs in the QTLs region was mainly for demethylation (33.3%-40.0%),which was higher than that of the average variation frequency of whole-genome.(3)Further analysis on 51 pairs of primers based on two retrotransposons (Houba and Osr14) demonstrated that there was higher level of frequency of DNA methylation and lower frequency of methylation pattern changes.These results suggested that the extensive DNA methylation level and pattern changes have been happened during the interspecific hybridization and alien gene introgression,which would provide useful information for efficiently exploiting and using the interesting wild genes for rice enhancement.