Abstract The present study was undertaken to evaluate the fatty acid composition, conjugated linoleic acid (CLA) and cholesterol contents in the milk from three yak breeds, Maiwa yak (n = 24), Zhongdian yak (n = 16) and Gannan yak (n = 16) and two yak crossbreeds, Maiwa yak × yellow cattle (n = 16) and Gannan yak × yellow cattle (n = 16). Statistically significant differences (P 0.05) among the three yak breeds. However, the percentage of total polyunsaturated fatty acids (PUFAs) in Zhongdian yak milk (4.82%) was significantly higher (P 0.05%) between the two yak crossbreeds. The cholesterol contents in milk samples from yak breeds and yak crossbreeds were found to be in the range 12.32–16.17 mg/100 g. Cholesterol contents in milk samples from yak breeds and yak crossbreeds were positively correlated with their fat contents.