The intramuscular fat composition of ruminant meats influences the quality of the final product, which explains the increasing interest in assessing the fatty acid profile of meat from different production systems. In this study, it was hypothesized that there are breed- and diet-induced variations on lipid metabolism in the muscle, which may be, at least partially, modulated by the stearoyl-CoA desaturase (SCD) gene expression levels. Forty purebred young bulls from two phylogenetically distant autochthonous cattle breeds, Alentejana and Barrosã (n = 20 for each breed), were assigned to two different diets (low vs. high silage) and slaughtered at 18 months of age. Meat fatty acid composition, including the detailed conjugated linoleic acid (CLA) isomeric profile, was determined along with the SCD mRNA levels. Meat from Barrosã bulls fed the low silage diet was richer in monounsaturated fatty acids, CLA and trans fatty acids, when compared to that from Alentejana bulls. The meat content in polyunsaturated fatty acids was similar across experimental groups. Moderate positive correlations between the SCD mRNA levels and the products of this enzyme activity were found, although they were not reflected on the calculated desaturase indices. Overall, these findings highlight the importance of taking into account the genetic background while devising feeding strategies to manipulate beef fatty acid composition.