Abstract: The milk samples of two hundred and thirty-five lactating Holstein dairy cows in north China were selected. In order to avoid confounding effects of diet and season, Holstein cows were fed a single diet and milk sampled on the same day. The performance of lactating dairy cows and composition of the fatty acids in milk fat were measured to investigate the effect of parity on conjugated linoleic acid (CLA) in milk fat and △⁹-desaturase indexes from Holstein dairy cows.The results showed that the average content of cis-9, trans-11 CLA was 5.14 mg/g fatty acids in milk fat, and the variation range was 1.5-10.5 mg/g fatty acids. The average content of cis-9, trans-11 CLA of primiparous was 5.19 mg/g fatty acids in milk fat, while it was 5.11 mg/g fatty acids of multiparous, and there's no significant difference between them（P>0.05）. There was significant difference between the △⁹-desaturase index of cis-9 C14∶1 and cis-9 C16∶1 of primiparous and multiparous（P<0.05）, and there was no significant difference between the △⁹-desaturase index of cis-9 C18∶1 and cis-9, trans-11 CLA of primiparous and multiparous（P>0.05）.