The impact of fresh legume types or silage on the composition of milk fatty acids and transcription of enzymes involved in the synthesis of milk fat in cows was studied. Three groups of cows grazed high proportions of white clover, red clover and lucerne, respectively. A fourth group of cows was fed maize/grass silage. The cows grazing high proportions of legumes produced significantly more 18:1 trans-11, 18:2 cis9-trans11, 18:2 trans10-cis12 and 18:3 fatty acids than cows fed silage. White clover and lucerne grazing resulted in significantly lower output of 18:1 trans9 in milk than red clover grazing and maize/grass silages. Transcription of stearoyl-CoA desaturase (SCD) in mammary tissue was significantly increased by grazing high proportions of legume whereas fatty acid synthase and acetyl-CoA carboxylase were not affected by type of feeding. Furthermore, average milk fat globule diameter was correlated to daily milk fat yield but was not affected by feeding. Although the fresh forage affected the transcription of SCD in mammary tissue, the largest effects were on the trans11-based fatty acids. It is concluded that type of forage, i.e. fresh or silage, had a greater impact on rumen fermentation pattern than on transcription of enzymes involved in the synthesis of milk fat.