Circadian E-boxes in the promoters of mPer1, mPer2, mCry1 and mDBP play a key role in regulating rhythmic gene expression by recruiting the BMAL1/CLOCK heterodimer. However, each of these circadian E-boxes seems different from one another. In the mouse liver, BMAL1/CLOCK binds to E-boxes in the mPer1 and mPer2 promoters constantly but binds with dynamic daily changes to mCry1 and mDBP promoters. It is unclear whether DNA methylation of these circadian E-boxes and the surrounding CpG islands would affect protein/DNA interactions and lead to distinct binding features. In the present study, bisulfite sequencing analysis indicated that all E-boxes and their surrounding CpG islands were free from methylation, suggesting that DNA methylation does not determine distinct binding features. The methylation status of the E-box in the MAP kinase phosphatase 1 (MKP1) promoter was also examined. Our results indicated that DNA methylation does not regulate the tissue-specific clock control of MKP1.