ABSTRACT.- Nitrated fatty acids (NO2-FAs), specifically nitroalkenes, are gaining recognition for their anti-inflammatory and therapeutic properties. Bovine milk, a major dietary component, may serve as a significant source of these bioactive molecules, complementing its established nutritional profile. This study examined nitroalkene's presence in milk from different bovine strains and lactation stages. NO2-conjugated linoleic acid (NO2-cLA) was detected at concentrations up to 5 uM, comprising 2.7% of total cLA. The concentration of NO2-cLA varied slightly with the Holstein strain and lactation stage, with its free form predominantly observed, suggesting limited triglyceride incorporation. Further research into NO2-cLA's biological significance in milk is required. © 2024 Society of Dairy Technology.