Abstract: In order to detect the methylation status of enoyl-CoA hydratase short chain 1 （ECHS1）, the sequence fragments of ECHS1 was obtained for Holstein cows by homologous cloning. The number of methylation was firstly predicted, and then methylation status of the obtained sequence fragments was detected by method of sequencing of bisulfite conversion. The results showed that fragments of ECHS1 with the size of 459 bp were obtained. C＋G content was 53.88%. The fragments contained 28 CpGs islands and all individuals at sites 1, 2, 9, 11, 13, 14, 17, 22, 24 and 27 had been methylated.