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) in the proportions of individual milk fatty acids were observed among the three yak breeds. The proportion of saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) did not differ statistically ( 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 < 0.01) than in Maiwa (3.99%) and Gannan (3.68%) yak milks. The contents of α-linolenic acid (C18:3) and cis-9, trans-11 conjugated linoleic acid (CLA) were significantly different ( P < 0.05) among the three yak breeds. The total SFAs, MUFAs and PUFAs were not significantly different ( 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.