Abstract: In order to extract mitochondrial DNA of high-quality Haloxylon ammodendron and Haloxylon persicum, the etiolated seedlings of Haloxylon ammodendron and Haloxylon persicum were taken as experimental materials. This research had established a method of Nuclear-free DNA mitochondrion was firstly obtained by combining sucrose density gradient centrifugation and differential centrifugation together and using DNaseⅠ. Then after the mitochondrion was lysed by using 3% CTAB and its protein was removed by chloroform-isoamyl alcohol extraction, purified mitochondrial DNA was finally got. With A260/A280≈1.8, the result showed that the extracted DNA presented distinct bands and no streaking during the electrophoresis, demonstrating that it enjoys high quality and purity, and can be applied in further experiments concerning mitochondrion DNA. The establishment of this method has laid a foundation for study on mitochondrial genome of Haloxylon ammodendron and Haloxylon persicum.