An experiment was conducted with 12 lactating dairy ewes and 12 goats with the objective to determine whether, under the same dietary treatments, the differences in their fatty acid (FA) profile with emphasis on cis-9 trans-11 CLA milk fat content, are reflected in the transcript levels of genes involved in FA and cis-9, trans-11 CLA biosynthesis. The animals were fed with two diets (A, B) in different days of milk (DIM) due to the different milk yield, body weight etc, in order to have the same food intake and to avoid dietary effects. Diet A was fed to the animals on a group basis as it is traditionally used in practice, while diet B was chosen to avoid individual feed intake variation which is usually observed in group feeding. The results showed that there are significantly lower mRNA levels of acetyl-CoA carboxylase (ACC) in sheep mammary gland compared with those of goats, independently from the diet fed. The same trend was observed with the mRNA level of FA synthase (FAS), but the results were significant only for diet A. The mRNA level of lipoprotein lipase (LPL) in the mammary gland did not differ between sheep and goats fed with diet A. In addition, the concentration of cis-9 trans-11 CLA content was significantly higher in sheep milk fat compared with those of goats. This is in accordance with the significant higher levels on mRNA of stearoyl-CoA desaturase (SCD) which were observed in their mammary adipocytes of sheep compared with those of goats, independently of the fed diet (A or B). In conclusion, these findings demonstrate that the differences between sheep and goats, concerning cis-9, trans-11 CLA and FA milk fat content, under the same dietary treatments could be explained in part by the differences in mRNA of SCD and lipogenic genes in their mammary gland.