To obtain high-quality DNA suitable for methylation-sensitive amplification polymorphism (MSAP), analysis, the mature cotyledon and tissue cultured cotyledon of walnut cultivar‘Liaoning-1’were used as the testing materials in the research of DNA isolation by 3 methods (SDS, high salt CTAB and modified high salt CTAB) and the mature leaf tissue was taken as the control. The concentration and intactness of the extracted DNA were determined using a spectrometer and by electrophoresis in 0.8% (w/v) agarose gels against standard solutions of lambda DNA. High quality DNA was extracted from leaf tissues using the high salt CTAB method and the modified high salt CTAB method. No detectable DNA was extracted from the mature cotyledon and cultured cotyledon using SDS method. The DNA with high quality but little quantity was extracted from the mature cotyledon but not the extract cultured cotyledon using the high salt CTAB method. The DNA with high quantity and quality suitable for MSAP analysis from both mature cotyledon and cultured cotyledon was extracted using the modified high salt CTAB method. The DNA extraction method of walnut cotyledon suitable for MSAP analysis was established, which could lay foundation for downstream molecular biology experiment and provide a reference to the polysaccharide removal research in DNA extraction procedure.