The identification of higher fat content in ewe milk during lactation can help to improve the nutritional value and quality of the derived dairy products. In this study, we characterized fatty acids from the wool of Suffolk ewes at two time points during lactation and assessed whether they were related to milk fat content through discriminant analysis and, thus, could be potentially used to identify ewes with a high fat content. Eighty single-bearing Suffolk ewes of similar body weight, body score, and age were selected for this study. The overall fat contents of milk and wool were determined, as well as the fatty acids in the wool. The wool fat content was 1.14% on average. The proportions of wool fatty acids were 65.82% saturated, 21.70% monounsaturated, and 12.48% polyunsaturated fatty acids. The wool fatty acid concentrations of C18:1n9c, C18:2n6c, and C22:2 were higher in ewes whose milk had a high fat content at both time points. Moreover, the levels of these fatty acids were positively correlated with milk fat content. Discriminant analyses using C18:1n9c and C18:2n6c were the best candidates for the prediction of high milk fat content, with an accuracy of 87.50%. The wool fatty acids C18:1n9c and C18:2n6c could potentially be used to determine the milk fat content of ewes.