The aim of this study was to identify Longissimus lumborum (LL) muscle from lambs with high IMF content (≥3%) using wool FA. The FA composition was characterized in two sections of wool from Suffolk Down lambs, and its relationship with IMF content assessed. One hundred and forty lambs of similar body weight, body condition score, and age were selected. IMF content from LL muscle, and the wool fat and FA composition of the wool were determined. The proportions of FAs in the wool of lambs with low IMF were 51.96 ± 7.3% of saturated, 31.96 ± 7.7% polyunsaturated, and 16.08 ± 2.3% monounsaturated. Similar proportions of FAs were observed in wool samples of lambs with high IMF. Significant differences were found only in the SFA proportion between groups (p < 0.05). The predominant FAs in the wool samples were C16:0, C17:0, C18:0, C18:1n9c and C18:2n6c, representing over 60% of total FA methyl esters. Correlations were found between the wool FAs C16:1, C17:0, C18:0, C18:1n9c, C24:0, and C22:6n3 in wool samples divided into two sections. Discriminant analysis identified SFAs, particularly FAs C22:0 and C24:0, as potential candidate for predicting lambs with high IMF content with an accuracy of over 92%. These results provide to our knowledge evidence that IMF content could potentially be determined using wool FAs as a non-invasive method.