The composition of Sn-2 fatty acids in yak milk fat was determined by gas chromatograph and TLC. The Sn-2 fatty acid composition of yak milk fat at different altitudes was studied to determine the effect of different altitudes on triglyceride structure. The results showed that there were 12 kinds of total fatty acids of triglyceride in Qinghai yak milk fat, and the highest contents were palmitic acid (31.35%～36.48%), oleic acid (24.91%～30.99%) and stearic acid (9.82%～13.87%). The composition of Sn-2 fatty acids in Qinghai yak milk fat was as follows: hexanoic acid (0.66%～1.02%), decanoic acid (2.46%～3.98%), lauric acid (2.43%～4.88%), tetradecanoic acid (14.66%～20.68%), pentadecanoic acid (1.19%～1.76%), palmitoic acid (39.91%～46.23%), stearic acid (4.75%～8.31%), oleic acid (17.42%～20.81%), linoleic acid (0.00%～1.72%), and linolenic acid (0.00%～1.71%). The significance analysis showed that the proportion of saturated fatty acids (SFA) binding to the Sn-2 of yak milk triglycerides increased gradually with the elevation, then decreased significantly; the proportion of unsaturated fatty acids (UFA) decreased gradually at first, then decreased gradually and then increased significantly.