High pressure homogenization (HPH) is a novel technology that promotes fat globule size reduction and microbial inactivation, but little research exists on the fate of milk fat lipids. This work studied the effect of HPH (0-350 MPa) of raw cow, goat and ewe milks on the fatty acid total content and profile to elucidate whether this technology has a major impact on the lipid fraction of milk and especially on CLA isomers. Fatty acids in processed milks were determined by GC-FID and CLA isomers by Ag+-HPLC. Our results indicate that the total amount of fat extracted from the milk samples decreased as the homogenization pressure increased, whereas no significant differences were found in the fatty acid composition, especially in the PUFA and CLA isomers profile of raw milk treated by HPH process up to 350 MPa. Industrial relevance: The absent of significant modifications of the fatty acids content and CLA isomers profile in milk by using high-pressure homogenization is relevant in the development of nonthermal technologies able to pasteurize/sterilize foods, without the organoleptic, functional, and chemical alterations associated to thermal processing.