Objective To establish a quantitative method to examine the methylation level of CpG island located in the intron 1 of mouse α-synuclein, and analyze the difference of methylation in various brain regions. Methods CpG island in the intron 1 of mouse α-synuclein was identified using an online software MSPprimer. Pyrosequencing assay was designed using PyroMark assay design 2.0. DNA methylation was quantified in mouse hippocampus, cortex, striatum and midbrain using pyrosequencing assay. Results There was a CpG island in the intron 1 of mouse α-synuclein. Pyrosequencing assay measuring methylation level of this CpG island was optimized. It indicated that all of the brain regions were hypomethylated with a methylation rate of 2% or less. Conclusion DNA methylation does not play a major role in the transcription regulation of mouse α-synuclein in the brain.